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Abstract:

The present invention provides placental stem cells and placental stem
cell populations, and methods of culturing, proliferating and expanding
the same. The invention also provides methods of differentiating the
placental stem cells. The invention further provides methods of using the
placental stem cells in assays and for transplanting.

Claims:

1. A method of producing a cell population comprising identifying
placental cells that adhere to a substrate and:express CD73, CD105, and
CD200; orexpress CD200 and OCT-4;

3. The method of claim 1, wherein said identifying is accomplished using
an antibody.

4. The method of claim 1, wherein said identifying is accomplished using
flow cytometry.

5. The method of claim 1, wherein said isolating is accomplished using
magnetic beads.

6. The method of claim 1, wherein said isolating is accomplished by
fluorescence-activated cell sorting.

7. The method of claim 1, wherein said cell population is expanded after
said isolating.

8. A method of producing a stem cell line, comprising transforming a stem
cell with a DNA sequence that encodes a growth-promoting protein; and
exposing said stem cell to conditions that promote production of said
growth-promoting protein.

15. The method of claim 14, wherein said adherent placental cell expresses
five or more of said genes at a detectably higher level than a bone
marrow-derived mesenchymal stem cell that has undergone the same number
of passages in culture as said placental stem cell.

16. The method of claim 14, wherein said adherent placental cell expresses
ten or more of said genes at a detectably higher level than a bone
marrow-derived mesenchymal stem cell that has undergone the same number
of passages in culture as said placental stem cell.

17. The method of claim 14, wherein said adherent placental cell expresses
twenty or more of said genes at a detectably higher level than a bone
marrow-derived mesenchymal stem cell that has undergone the same number
of passages in culture as said placental stem cell.

18. The method of claim 14, wherein said adherent placental cell expresses
each of said genes at a detectably higher level than a bone
marrow-derived mesenchymal stem cell that has undergone the same number
of passages in culture as said placental stem cell.

[0003]Human stem cells are totipotential or pluripotential precursor cells
capable of generating a variety of mature human cell lineages. Evidence
exists that demonstrates that stem cells can be employed to repopulate
many, if not all, tissues and restore physiologic and anatomic
functionality.

[0006]The invention first provides isolated stem cells, and cell
populations comprising such stem cells, wherein the stem cells are
present in, and isolatable from placental tissue (e.g., amnion, chorion,
placental cotyledons, etc.) The placental stem cells exhibit one or more
characteristics of a stem cell (e.g., exhibit markers associated with
stem cells, replicate at least 10-20 times in culture in an
undifferentiated state, differentiate into adult cells representative of
the three germ layers, etc.), and can adhere to a tissue culture
substrate (e.g., tissue culture plastic such as the surface of a tissue
culture dish or multiwell plate).

[0007]In one embodiment, the invention provides an isolated placental stem
cell that is CD200+ or HLA-G+. In a specific embodiment, said
cell is CD200+ and HLA-G+. In a specific embodiment, said stem
cell is CD73 and CD105+. In another specific embodiment, said stem
cell is CD34, CD38 or CD45. In another specific embodiment, said stem
cell is CD34-, CD38- and CD45-. In another specific
embodiment, said stem cell is CD34-, CD38-, CD45-,
CD73+ and CD105+. In another specific embodiment, said stem
cell facilitates the formation of one or more embryoid-like bodies from a
population of isolated placental cells comprising placental stem cells
when said population is cultured under conditions that allow formation of
embryoid-like bodies.

[0008]In another embodiment, the invention provides a population of
isolated placental cells comprising, e.g., that is enriched for,
CD200-, HLA-G+ stem cells. In various embodiments, at least
10%, at least 20%, at least 30%, at least 40%, at least 50% at least 60%,
at least 70%, at least 80%, at least 90%, or at least 95% or more of said
isolated placental cells are CD200+, HLA-G+ stem cells. In a
specific embodiment of the above populations, said stem cells are
CD73+ and CD105+. In another specific embodiment, said stem
cells are CD34-, CD38- or CD45-. In a more specific
embodiment, said stem cells are CD34-, CD38-, CD45-,
CD73+ and CD105+. In other specific embodiments, said
population has been expanded, e.g., passaged at least once, at least
three times, at least five times, at least 10 times, at least 15 times,
or at least 20 times. In another specific embodiment, said population
forms one or more embryoid-like bodies when cultured under conditions
that allow formation of embryoid-like bodies.

[0009]In another embodiment, the invention provides an isolated stem cell
that is CD73+, CD105+, and CD200+. In a specific
embodiment, said stem cell is HLA-G+. In another specific
embodiment, said stem cell is CD34-, CD38- or CD45-. In
another specific embodiment, said stem cell is CD34-, CD38- and
CD45-. In a more specific embodiment, said stem cell is CD34-,
CD38-, CD45-, and HLA-G+. In another specific embodiment,
said stem cell facilitates development of one or more embryoid-like
bodies from a population of isolated placental cells comprising the stem
cell when said population is cultured under conditions that allow
formation of embryoid-like bodies.

[0010]In another embodiment, the invention provides a population of
isolated placental cells comprising, e.g., that is enriched for,
CD73+, CD105+, CD200+ stem cells. In various embodiments,
at least 10%, at least 20%, at least 30%, at least 40%, at least 50% at
least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of
said isolated placental cells are CD73+, CD105+, CD200+
stem cells. In a specific embodiment of said populations, said stem cells
are HLA-G+. In another specific embodiment, said stem cells are
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cells are CD34-, CD38- and CD45-. In a more
specific embodiment, said stem cells are CD34-, CD38-,
CD45-, and HLA-G+. In other specific embodiments, said
population has been expanded, for example, passaged at least once, at
least three times, at least five times, at least 10 times, at least 15
times, or at least 20 times. In another specific embodiment, said
population forms one or more embryoid-like bodies in culture under
conditions that allow formation of embryoid-like bodies.

[0011]The invention also provides an isolated stem cell that is
CD200+ and OCT-4+. In a specific embodiment, the stem cell is
CD73+ and CD105+. In another specific embodiment, said stem
cell is HLA-G+. In another specific embodiment, said stem cell is
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cell is CD34-, CD38- and CD45-. In a more
specific embodiment, said stem cell is CD34-, CD38-,
CD45-, CD73+, CD105- and HLA-G+. In another specific
embodiment, said stem cell facilitates the formation of one or more
embryoid-like bodies from a population of isolated placental cells
comprising placental stem cells when said population is cultured under
conditions that allow formation of embryoid-like bodies.

[0012]In another embodiment, the invention provides a population of
isolated cells comprising, e.g., that is enriched for, CD200-,
OCT-4+ stem cells. In various embodiments, at least 10%, at least
20%, at least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said isolated placental
cells are CD200+, OCT-4+ stem cells. In a specific embodiment
of the above populations, said stem cells are CD73+ and CD105+.
In another specific embodiment, said stem cells are HLA-G+. In
another specific embodiment, said stem cells are CD34-, CD38and CD45-. In a more specific embodiment, said stem cells are
CD34-, CD38-, CD45-, CD73+, CD105+ and
HLA-G+. In other specific embodiments, said population has been
expanded, for example, has been passaged at least once, at least three
times, at least five times, at least 10 times, at least 15 times, or at
least 20 times. In another specific embodiment, said population forms one
or more embryoid-like bodies when cultured under conditions that allow
the formation of embryoid-like bodies.

[0013]In another embodiment, the invention provides an isolated stem cell
that is CD73+ and CD105+ and which facilitates the formation of
one or more embryoid-like bodies in a population of isolated placental
cells comprising said stem cell when said population is cultured under
conditions that allow formation of embryoid-like bodies. In a specific
embodiment, said stem cell is CD34-, CD38- or CD45-. In
another specific embodiment, said stem cell is CD34-, CD38- and
CD45-. In another specific embodiment, said stem cell is OCT4+.
In a more specific embodiment, said stem cell is OCT4+, CD34-,
CD38-and CD45-.

[0014]The invention further provides a population of isolated placental
cells comprising, e.g., that is enriched for, CD73+, CD105+
stem cells, wherein said population forms one or more embryoid-like
bodies under conditions that allow formation of embryoid-like bodies. In
various embodiments, at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at least 90%,
or at least 95% of said isolated placental cells are CD73-,
CD105+ stem cells. In a specific embodiment of the above
populations, said stem cells are CD34-, CD38- or CD45-. In
another specific embodiment, said stem cells are CD34-, CD38and CD45-. In another specific embodiment, said stem cells are
OCT-4-. In a more specific embodiment, said stem cells are
OCT-4+, CD34-, CD38- and CD45-. In other specific
embodiments, said population has been expanded, for example, has been
passaged at least once, at least three times, at least five times, at
least 10 times, at least 15 times, or at least 20 times.

[0015]The invention further provides an isolated stem cell that is
CD73+, CD105+and HLA-G+. In a specific embodiment, said
stem cell is CD34-, CD38- or CD45-. In another specific
embodiment, said stem cell is CD34-, CD38- and CD45-. In
another specific embodiment, said stem cell is OCT-4+. In another
specific embodiment, said stem cell is CD200+. In a more specific
embodiment, said stem cell is CD34-, CD38-, CD45-,
OCT-4+ and CD200+. In another specific embodiment, said stem
cell facilitates the formation of one or more embryoid-like bodies from a
population of isolated placental cells comprising placental stem cells in
culture under conditions that allow formation of embryoid-like bodies.

[0016]The invention further provides a population of isolated placental
cells comprising, e.g., that is enriched for, CD73+, CD105+ and
HLA-G+ stem cells. In various embodiments, at least 10%, at least
20%, at least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said isolated placental
cells are CD73+, CD105+ and HLA-G+ stem cells. In a
specific embodiment of the above populations, said stem cells are
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cells are CD34-, CD38- and CD45-. In another
specific embodiment, said stem cells are OCT-4+. In another specific
embodiment, said stem cells are CD200+. In a more specific
embodiment, said stem cells are CD34, CD38, CD45, OCT-4- and
CD200+. In another specific embodiment, said population has been
expanded, for example, has been passaged at least once, at least three
times, at least five times, at least 10 times, at least 15 times, or at
least 20 times. In another specific embodiment, said population forms
embryoid-like bodies when cultured under conditions that allow the
formation of embryoid-like bodies.

[0017]The invention further provides an isolated stem cell that is
OCT-4+ and which facilitates formation of one or more embryoid-like
bodies in a population of isolated placental cells comprising said stem
cell when cultured under conditions that allow formation of embryoid-like
bodies. In a specific embodiment, said stem cell is CD73+ and
CD105+. In another specific embodiment, said stem cell is
CD34-, CD38-, or CD45-. In another specific embodiment,
said stem cell is CD200+. In a more specific embodiment, said stem
cell is CD73+, CD105-, CD200+, CD34-, CD38-, and
CD45-.

[0018]The invention also provides a population of isolated cells
comprising, e.g., that is enriched for, OCT-4-placental stem cells,
wherein said population forms one or more embryoid-like bodies when
cultured under conditions that allow the formation of embryoid-like
bodies. In various embodiments, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said isolated placental cells are
OCT4+ placental stem cells. In a specific embodiment of the above
populations, said stem cells are CD73+ and CD105+. In another
specific embodiment, said stem cells are CD34-, CD38-, or
CD45-. In another specific embodiment, said stem cells are
CD200+. In a more specific embodiment, said stem cells are
CD73+, CD105+, CD200+, CD34-, CD38-, and
CD45-. In another specific embodiment, said population has been
expanded, for example, passaged at least once, at least three times, at
least five times, at least 10 times, at least 15 times, or at least 20
times.

[0019]The invention further provides an isolated population of the
placental stem cells described herein that is produced according to a
method comprising perfusing a mammalian placenta that has been drained of
cord blood and perfused to remove residual blood; perfusing said placenta
with a perfusion solution; and collecting said perfusion solution,
wherein said perfusion solution after perfusion comprises a population of
placental cells that comprises placental stem cells; and isolating a
plurality of said placental stem cells from said population of cells. In
a specific embodiment, the perfusion solution is passed through both the
umbilical vein and umbilical arteries and collected after it exudes from
the placenta. In another specific embodiment, the perfusion solution is
passed through the umbilical vein and collected from the umbilical
arteries, or passed through the umbilical arteries and collected from the
umbilical vein.

[0020]The invention further provides an isolated population of the
placental stem cells described herein that is produced according to a
method comprising digesting placental tissue with a tissue-disrupting
enzyme to obtain a population of placental cells comprising placental
stem cells, and isolating a plurality of placental stem cells from the
remainder of said placental cells. In specific embodiments, said
placental tissue is a whole placenta, an amniotic membrane, chorion, a
combination of amnion and chorion, or a combination of any of the
foregoing. In other specific embodiment, the tissue-disrupting enzyme is
trypsin or collagenase.

[0025]The invention also provides compositions that comprise one or more
of the stem cells of the invention, wherein the stem cell has been
isolated from the placenta. Thus, the invention further provides a
composition comprising a stem cell, wherein said stem cell is CD200+
and HLA-G+. In a specific embodiment, said stem cell is CD73+
and CD105+. In another specific embodiment, said stem cell is
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cell is CD34-, CD38- and CD45-. In a more
specific embodiment, said stem cell is CD34-, CD38-,
CD45-, CD73+, CD105+, CD200+ and HLA-G+.

[0026]In another embodiment, the invention provides a composition
comprising a stem cell, wherein said stem cell is CD73-, CD105+
and CD200+. In a specific embodiment, said stem cell is HLA-G-.
In another specific embodiment, said stem cell is CD34-, CD38or CD45-. In another specific embodiment, said stem cell is
CD34-, CD38- and CD45-. In another specific embodiment,
said stem cell is CD34-, CD38-, CD45-, and HLA-G+.

[0027]In another embodiment, the invention provides a composition
comprising a stem cell, wherein said stem cell is CD200+ and
OCT-4+. In a specific embodiment, said stem cell is CD73+ and
CD105+. In another specific embodiment, said stem cell is
HLA-G+. In another specific embodiment, said stem cell is
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cell is CD34-, CD38- and CD45-. In another
specific embodiment, said stem cell is CD34-, CD38-,
CD45-, CD73+, CD105+, and HLA-G+.

[0028]In another embodiment, the invention provides a composition
comprising a stem cell that is CD73+ and CD105+, wherein said
stem cell facilitates formation of an embryoid-like body in a population
of isolated placental cells comprising said stem cell under conditions
that allow the formation of an embryoid-like body. In a specific
embodiment, said stem cell is CD34-, CD38- or CD45-. In
another specific embodiment, said stem cell is OCT-4+. In another
specific embodiment, said stem cell is CD200+. In another specific
embodiment, said stem cell is OCT-4+, CD200+, CD34, CD38 and CD45.

[0029]In yet another embodiment, the invention provides a composition
comprising a stem cell that is CD73+, CD105+ and HLA-G+.
In a specific embodiment, said stem cell is CD34-, CD38- or
CD45-. In another specific embodiment, said stem cell is
OCT-4+. In another specific embodiment, said stem cell is
CD200+. In another specific embodiment, said stem cell is OCT-4+,
CD200+, CD34-, CD38- and CD45-.

[0030]In another embodiment, the invention provides a composition
comprising a stem cell that is OCT-4-, wherein said stem cell
facilitates formation of an embryoid-like body in a population of
isolated placental cells comprising said stem cell under conditions that
allow the formation of an embryoid-like body. In a specific embodiment,
said stem cell is CD73+ and CD105+. In another specific
embodiment, said stem cell is CD34-, CD38- and CD45-. In
another specific embodiment, said stem cell is CD200+. In another
specific embodiment, said stem cell is CD73+, CD105+,
CD200+, CD34-, CD38- and CD45-.

[0032]In another specific embodiment, any of the foregoing compositions
comprises a matrix. In a more specific embodiment, said matrix is a
three-dimensional scaffold. In another more specific embodiment, said
matrix comprises collagen, gelatin, laminin, fibronectin, pectin,
ornithine, or vitronectin. In another more specific embodiment, the
matrix is an amniotic membrane or an amniotic membrane-derived
biomaterial. In another more specific embodiment, said matrix comprises
an extracellular membrane protein. In another more specific embodiment,
said matrix comprises a synthetic compound. In another more specific
embodiment, said matrix comprises a bioactive compound. In another more
specific embodiment, said bioactive compound is a growth factor,
cytokine, antibody, or organic molecule of less than 5,000 daltons.

[0033]In another embodiment, the invention further provides a composition
comprising medium conditioned by any of the foregoing stem cells, or any
of the foregoing stem cell populations. In a specific embodiment, any
such composition comprises a stem cell that is not derived from a
placenta. In a more specific embodiment, said stem cell is an embryonic
stem cell. In another more specific embodiment, said stem cell is a
mesenchymal stem cell. In another more specific embodiment, said stem
cell is a bone marrow-derived stem cell. In another more specific
embodiment, said stem cell is a hematopoietic progenitor cell. In another
more specific embodiment, said stem cell is a somatic stem cell. In an
even more specific embodiment, said somatic stem cell is a neural stem
cell, a hepatic stem cell, a pancreatic stem cell, an endothelial stem
cell, a cardiac stem cell, or a muscle stem cell.

[0034]The invention also provides methods for producing populations of
stem cells derived from mammalian placenta. In one embodiment, for
example, the invention provides a method of producing a cell population
comprising selecting cells that (a) adhere to a substrate, and (b)
express CD200 and HLA-G; and isolating said cells from other cells to
form a cell population. In another embodiment, the invention provides a
method of producing a cell population, comprising selecting cells that
(a) adhere to a substrate, and (b) express CD73, CD105, and CD200; and
isolating said cells from other cells to form a cell population. In
another embodiment, the invention provides a method of producing a cell
population, comprising selecting cells that (a) adhere to a substrate and
(b) express CD200 and OCT-4; and isolating said cells from other cells to
form a cell population. In yet another embodiment, the invention provides
a method of producing a cell population, comprising selecting cells that
(a) adhere to a substrate, (b) express CD73 and CD105, and (c) facilitate
the formation of one or more embryoid-like bodies when cultured with a
population of placental cells under conditions that allow for the
formation of embryoid-like bodies; and isolating said cells from other
cells to form a cell population. In another embodiment, the invention
provides a method of producing a cell population, comprising selecting
cells that (a) adhere to a substrate, and (b) express CD73, CD105 and
HLA-G; and isolating said cells from other cells to form a cell
population. The invention also provides a method of producing a cell
population, comprising selecting cells that (a) adhere to a substrate,
(b) express OCT-4, and (c) facilitate the formation of one or more
embryoid-like bodies when cultured with a population of placental cells
under conditions that allow for the formation of embryoid-like bodies;
and isolating said cells from other cells to form a cell population. In a
specific embodiment of any of the foregoing methods, said substrate
comprises fibronectin. In another specific embodiment, the methods
comprise selecting cells that express ABC-p. In another specific
embodiment, the methods comprise selecting cells exhibiting at least one
characteristic specific to a mesenchymal stem cell. In a more specific
embodiment, said characteristic specific to a mesenchymal stem cell is
expression of CD29, expression of CD44, expression of CD90, or expression
of a combination of the foregoing. In another specific embodiment of the
methods, said selecting is accomplished using an antibody. In another
specific embodiment, said selecting is accomplished using flow cytometry.
In another specific embodiment, said selecting is accomplished using
magnetic beads. In another specific embodiment, said selecting is
accomplished by fluorescence-activated cell sorting. In another specific
embodiment of the above methods, said cell population is expanded.

[0035]The invention also provides a method of producing a stem cell line,
comprising transforming a stem cell with a DNA sequence that encodes a
growth-promoting protein; and exposing said stem cell to conditions that
promote production of said growth-promoting protein. In a specific
embodiment, said growth-promoting protein is v-myc, N-myc, c-myc, p53,
SV40 large T antigen, polyoma large T antigen, E1a adenovirus or human
papillomavirus E7 protein. In a more specific embodiment, said DNA
sequence is regulatable. In more specific embodiment, said DNA sequence
is regulatable by tetracycline. In another specific embodiment, said
growth-promoting protein has a regulatable activity. In another specific
embodiment, said growth-promoting protein is a temperature-sensitive
mutant.

[0036]The invention further provides cryopreserved stem cell populations.
For example, the invention provides a population of CD200+,
HLA-G+ stem cells, wherein said cells have been cryopreserved, and
wherein said population is contained within a container. The invention
also provides a population of CD73+, CD105+, CD200+ stem
cells, wherein said stem cells have been cryopreserved, and wherein said
population is contained within a container. The invention also provides a
population of CD200+, OCT-4+ stem cells, wherein said stem
cells have been cryopreserved, and wherein said population is contained
within a container. The invention also provides a population of
CD73+, CD105+ stem cells, wherein said cells have been
cryopreserved, and wherein said population is contained within a
container, and wherein said stem cells facilitate the formation of one or
more embryoid-like bodies when cultured with a population of placental
cells under conditions that allow for the formation of embryoid-like
bodies. The invention further provides a population of CD73+, CD105,
HLA-G+ stem cells, wherein said cells have been cryopreserved, and
wherein said population is contained within a container. The invention
also provides a population of OCT-4+ stem cells, wherein said cells
have been cryopreserved, wherein said population is contained within a
container, and wherein said stem cells facilitate the formation of one or
more embryoid-like bodies when cultured with a population of placental
cells under conditions that allow for the formation of embryoid-like
bodies. In a specific embodiment of any of the foregoing cryopreserved
populations, said container is a bag. In various specific embodiments,
said population comprises about, at least, or at most 1×106
said stem cells, 5×106 said stem cells, 1×107 said
stem cells, 5×107 said stem cells, 1×108 said stem
cells, 5×108 said stem cells, 1×109 said stem
cells, 5×109 said stem cells, or 1×1010 said stem
cells. In other specific embodiments of any of the foregoing
cryopreserved populations, said stem cells have been passaged about, at
least, or no more than 5 times, no more than 10 times, no more than 15
times, or no more than 20 times. In another specific embodiment of any of
the foregoing cryopreserved populations, said stem cells have been
expanded within said container.

3.1 Definitions

[0037]As used herein, the term "SH2" refers to an antibody that binds an
epitope on the marker CD 105. Thus, cells that are referred to as
SH2+ are CD105+.

[0038]As used herein, the terms "SH3" and SH4" refer to antibodies that
bind epitopes present on the marker CD73. Thus, cells that are referred
to as SH3+ and/or SH4+ are CD73+.

[0039]As used herein, the term "isolated stem cell" means a stem cell that
is substantially separated from other, non-stem cells of the tissue,
e.g., placenta, from which the stem cell is derived. A stem cell is
"isolated" if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of
the non-stem cells with which the stem cell is naturally associated, or
stem cells displaying a different marker profile, are removed from the
stem cell, e.g., during collection and/or culture of the stem cell.

[0040]As used herein, the term "population of isolated cells" means a
population of cells that is substantially separated from other cells of
the tissue, e.g., placenta, from which the population of cells is
derived. A stem cell is "isolated" if at least 50%, 60%, 70%, 80%, 90%,
95%, or at least 99% of the cells with which the population of cells, or
cells from which the population of cells is derived, is naturally
associated, i.e., stem cells displaying a different marker profile, are
removed from the stem cell, e.g., during collection and/or culture of the
stem cell.

[0041]As used herein, the term "placental stem cell" refers to a stem cell
or progenitor cell that is derived from a mammalian placenta, regardless
of morphology, cell surface markers, or the number of passages after a
primary culture. The term "placental stem cell" as used herein does not,
however, refer to a trophoblast. A cell is considered a "stem cell" if
the cell retains at least one attribute of a stem cell, e.g., a marker or
gene expression profile associated with one or more types of stem cells;
the ability to replicate at least 10-40 times in culture, the ability to
differentiate into cells of all three germ layers; the lack of adult
(i.e., differentiated) cell characteristics, or the like. The terms
"placental stem cell" and "placenta-derived stem cell" may be used
interchangeably.

[0042]As used herein, a stem cell is "positive" for a particular marker
when that marker is detectable above background. For example, a placental
stem cell is positive for, e.g., CD73 because CD73 is detectable on
placental stem cells in an amount detectably greater than background (in
comparison to, e.g., an isotype control). A cell is also positive for a
marker when that marker can be used to distinguish the cell from at least
one other cell type, or can be used to select or isolate the cell when
present or expressed by the cell. In the context of, e.g.,
antibody-mediated detection, "positive," as an indication a particular
cell surface marker is present, means that the marker is detectable using
an antibody, e.g., a fluorescently-labeled antibody, specific for that
marker; "positive" also means that a cell bears that marker in a amount
that produces a signal, e.g., in a cytometer, that is detectably above
background. For example, a cell is "CD200+" where the cell is
detectably labeled with an antibody specific to CD200, and the signal
from the antibody is detectably higher than a control (e.g., background).
Conversely, "negative" in the same context means that the cell surface
marker is not detectable using an antibody specific for that marker
compared to background. For example, a cell is "CD34-" where the
cell is not detectably labeled with an antibody specific to CD34. Unless
otherwise noted herein, cluster of differentiation ("CD") markers are
detected using antibodies. OCT-4 is determined to be present, and a cell
is "OCT-4+" if OCT-4 is detectable using RT-PCR.

[0052]FIG. 10: Culture time courses for amnion/chorion (AC), umbilical
cord (UC), bone marrow-derived stem cell (BM-MSC) and human dermal
fibroblast (NHDF) cell lines used in this study. All cultures were grown
and propagated using the same seeding and passage densities. Circles
indicate which cultures were used for RNA isolation. Late cultures were
harvested just prior to senescence. Two UC cultures were harvested at 38
doublings (UC-38) to compare the effect of trypsinization on gene
expression. All other cultures were lysed directly in their culture
flasks prior to RNA isolation.

[0053]FIG. 11: Line plot of relative expression levels of 8215 genes in
amnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell
(BM-MSC) and human dermal fibroblast (DF) cells. The number associated
with each cell line designation on the X-axis indicates the number of
days the cell line was cultured prior to evaluation of gene expression
levels. The chart was generated from RNA expression data analyzed by
GeneSpring software. AC-03 was used as the selected condition.

[0054]FIG. 12: Subset of the all genes list showing genes over-expressed
≧6-fold in AC-03 for amnion/chorion (AC), umbilical cord (UC),
bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast (DF)
cells. The number associated with each cell line designation on the
X-axis indicates the number of days the cell line was cultured prior to
evaluation of gene expression levels. The chart was generated from RNA
expression data analyzed by GeneSpring software. AC-03 was used as the
selected condition.

[0055]FIG. 13: Placental stem cell-specific or umbilical cord stem
cell-specific genes found by fold change filtering for amnion/chorion
(AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) and
human dermal fibroblast (DF) cells. The number associated with each cell
line designation on the X-axis indicates the number of days the cell line
was cultured prior to evaluation of gene expression levels. The chart was
generated from RNA expression data analyzed by GeneSpring software. AC-03
was used as the selected condition.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Placental Stem Cells and Placental Stem Cell Populations

[0056]Placental stem cells are stem cells, obtainable from a placenta or
part thereof, that adhere to a tissue culture substrate and have the
capacity to differentiate into non-placental cell types. Placental stem
cells can be either fetal or maternal in origin (that is, can have the
genotype of either the fetus or mother, respectively). Preferably, the
placental stem cells and placental stem cell populations of the invention
are fetal in origin. Populations of placental stem cells, or populations
of cells comprising placental stem cells, can comprise placental stem
cells that are solely fetal or maternal in origin, or can comprise a
mixed population of placental stem cells of both fetal and maternal
origin. The placental stem cells, and populations of cells comprising the
placental stem cells, can be identified and selected by the
morphological, marker, and culture characteristic discussed below.

5.1.1 Physical and Morphological Characteristics

[0057]The placental stem cells of the present invention, when cultured in
primary cultures or in cell culture, adhere to the tissue culture
substrate, e.g., tissue culture container surface (e.g., tissue culture
plastic). Placental stem cells in culture assume a generally
fibroblastoid, stellate appearance, with a number of cyotplasmic
processes extending from the central cell body. The placental stem cells
are, however, morphologically differentiable from fibroblasts cultured
under the same conditions, as the placental stem cells exhibit a greater
number of such processes than do fibroblasts. Morphologically, placental
stem cells are also differentiable from hematopoietic stem cells, which
generally assume a more rounded, or cobblestone, morphology in culture.

[0058]5.1.2 Cell Surface, Molecular and Genetic Markers

[0059]Placental stem cells of the present invention, and populations of
placental stem cells, express a plurality of markers that can be used to
identify and/or isolate the stem cells, or populations of cells that
comprise the stem cells. The placental stem cells, and stem cell
populations of the invention (that is, two or more placental stem cells)
include stem cells and stem cell-containing cell populations obtained
directly from the placenta, or any part thereof (e.g., amnion, chorion,
placental cotyledons, and the like). Placental stem cell populations also
includes populations of (that is, two or more) placental stem cells in
culture, and a population in a container, e.g., a bag. Placental stem
cells are not, however, trophoblasts.

[0060]The placental stem cells of the invention generally express the
markers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do not express CD34,
CD38, or CD45. Placental stem cells can also express HLA-ABC (MHC-1) and
HLA-DR. These markers can be used to identify placental stem cells, and
to distinguish placental stem cells from other stem cell types. Because
the placental stem cells can express CD73 and CD105, they can have
mesenchymal stem cell-like characteristics. However, because the
placental stem cells can express CD200 and HLA-G, a fetal-specific
marker, they can be distinguished from mesenchymal stem cells, e.g., bone
marrow-derived mesenchymal stem cells, which express neither CD200 nor
HLA-G. In the same manner, the lack of expression of CD34, CD38 and/or
CD45 identifies the placental stem cells as non-hematopoietic stem cells.

[0061]Thus, in one embodiment, the invention provides an isolated stem
cell that is CD200+ or HLA-G+. In a specific embodiment, said
stem cell is a placental stem cell. In a specific embodiment, the stem
cell is CD200+ and HLA-G+. In a specific embodiment, said stem
cell is CD73+ and CD105+. In another specific embodiment, said
stem cell is CD34-, CD38- or CD45-. In another specific
embodiment, said stem cell is CD34-, CD38- and CD45-. In
another specific embodiment, said stem cell is CD34-, CD38-,
CD45-, CD73+ and CD105+. In another specific embodiment,
said CD200+ or HLA-G+ stem cell facilitates the formation of
embryoid-like bodies in a population of placental cells comprising the
stem cells, under conditions that allow the formation of embryoid-like
bodies. In another specific embodiment, said placental stem cell is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said placental stem cell is isolated away from
placental stem cells that do not display these markers.

[0062]In another embodiment, the invention also provides a method of
selecting a placental stem cell from a plurality of placental cells,
comprising selecting a CD200+ or HLA-G+ placental cell, whereby
said cell is a placental stem cell. In a specific embodiment, said
selecting comprises selecting a placental cell that is both CD200+
and HLA-G+. In a specific embodiment, said selecting comprises
selecting a placental cell that is also CD73+ and CD105+. In
another specific embodiment, said selecting comprises selecting a
placental cell that is also CD34-, CD38- or CD45-. In
another specific embodiment, said selecting comprises selecting a
placental cell that is also CD34-, CD38- and CD45-. In
another specific embodiment, said selecting comprises selecting a
placental cell that is also CD34-, CD38-, CD45-,
CD73+ and CD105-. In another specific embodiment, said
selecting comprises selecting a placental cell that also facilitates the
formation of embryoid-like bodies in a population of placental cells
comprising the stem cells, under conditions that allow the formation of
embryoid-like bodies.

[0063]In another embodiment, the invention provides an isolated population
of cells comprising, e.g., that is enriched for, CD200-, HLA-G+
stem cells. In a specific embodiment, said population is a population of
placental cells. In various embodiments, at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, or
at least about 60% of said cells are CD200+, HLA-G+ stem cells.
Preferably, at least about 70% of said cells are CD200+, HLA-G+
stem cells. More preferably, at least about 90%, 95%, or 99% of said
cells are CD200+, HLA-G+ stem cells. In a specific embodiment
of the isolated populations, said stem cells are also CD73- and
CD105+. In another specific embodiment, said stem cells are also
CD34-, CD38- or CD45-. In a more specific embodiment, said
stem cells are also CD34-, CD38-, CD45-, CD73+ and
CD105+. In another embodiment, said isolated population produces one
or more embryoid-like bodies when cultured under conditions that allow
the formation of embryoid-like bodies. In another specific embodiment,
said population of placental stem cells is isolated away from placental
cells that are not stem cells. In another specific embodiment, said
population of placental stem cells is isolated away from placental stem
cells that do not display these markers.

[0064]In another embodiment, the invention also provides a method of
selecting a placental stem cell population from a plurality of placental
cells, comprising selecting a population of placental cells wherein at
least about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50% at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or at least about 95% of said cells are
CD200+, HLA-G+ stem cells. In a specific embodiment, said
selecting comprises selecting stem cells that are also CD73+ and
CD105+. In another specific embodiment, said selecting comprises
selecting stem cells that are also CD34-, CD38- or CD45-.
In another specific embodiment, said selecting comprises selecting stem
cells that are also CD34-, CD38-, CD45-, CD73+ and
CD105+. In another specific embodiment, said selecting also
comprises selecting a population of placental stem cells that forms one
or more embryoid-like bodies when cultured under conditions that allow
the formation of embryoid-like bodies.

[0065]In another embodiment, the invention provides an isolated stem cell
that is CD73+, CD105+, and CD200+. In an specific
embodiment, said isolated stem cell is an isolated placental stem cell.
In another specific embodiment, said stem cell is HLA-G+. In another
specific embodiment, said stem cell is CD34-, CD38- or
CD45-. In another specific embodiment, said stem cell is CD34-,
CD38- and CD45-. In a more specific embodiment, said stem cell
is CD34-, CD38-, CD45-, and HLA-G+. In another
specific embodiment, the isolated CD73+, CD105+, and
CD200+ stem cell facilitates the formation of one or more
embryoid-like bodies in a population of placental cells comprising the
stem cell, when the population is cultured under conditions that allow
the formation of embryoid-like bodies. In another specific embodiment,
said placental stem cell is isolated away from placental cells that are
not stem cells. In another specific embodiment, said placental stem cell
is isolated away from placental stem cells that do not display these
markers.

[0066]In another embodiment, the invention also provides a method of
selecting a placental stem cell from a plurality of placental cells,
comprising selecting a CD73+, CD105+, and CD200+ placental
cell, whereby said cell is a placental stem cell. In a specific
embodiment, said selecting comprises selecting a placental cell that is
also HLA-G+. In another specific embodiment, said selecting
comprises selecting a placental cell that is also CD34-, CD38or CD45-. In another specific embodiment, said selecting comprises
selecting a placental cell that is also CD34-, CD38- and
CD45-. In another specific embodiment, said selecting comprises
selecting a placental cell that is also CD34, CD38, CD45, and
HLA-G+. In another specific embodiment, said selecting additionally
comprises selecting a CD73+, CD105+, and CD200+ stem cell
that facilitates the formation of one or more embryoid-like bodies in a
population of placental cells comprising the stem cell, when the
population is cultured under conditions that facilitate formation of
embryoid-like bodies.

[0067]In another embodiment, the invention provides an isolated population
of cells comprising, e.g., that is enriched for, CD73+, CD105+,
CD200+ stem cells. In a specific embodiment, said stem cells are
placental stem cells. In various embodiments, at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least about
50%, or at least about 60% of said cells are CD73+, CD105+,
CD200+ stem cells. In another embodiment, at least about 70% of said
cells in said population of cells are CD73+, CD105+,
CD200+ stem cells. In another embodiment, at least about 90%, 95% or
99% of said cells in said population of cells are CD73+,
CD105-, CD200+ stem cells. In a specific embodiment of said
populations, said stem cells are HLA-G+. In another specific
embodiment, said stem cells are CD34-, CD38- or CD45-. In
another specific embodiment, said stem cells are CD34-, CD38and CD45-. In a more specific embodiment, said stem cells are
CD34-, CD38-, CD45-, and HLA-G+. In another specific
embodiment, said population of cells produces one or more embryoid-like
bodies when cultured under conditions that allow the formation of
embryoid-like bodies. In another specific embodiment, said population of
placental stem cells is isolated away from placental cells that are not
stem cells. In another specific embodiment, said population of placental
stem cells is isolated away from placental stem cells that do not display
these characteristics.

[0068]In another embodiment, the invention also provides a method of
selecting a placental stem cell population from a plurality of placental
cells, comprising selecting a population of placental cells wherein at
least about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or at least about 95% of said cells are
CD73+, CD105+, CD200+ stem cells. In a specific
embodiment, said selecting comprises selecting stem cells that are also
HLA-G+. In another specific embodiment, said selecting comprises
selecting stem cells that are also CD34-, CD38- or CD45-.
In another specific embodiment, said selecting comprises selecting stem
cells that are also CD34-, CD38- and CD45-. In another
specific embodiment, said selecting comprises selecting stem cells that
are also CD34-, CD38-, CD45-, and HLA-G+. In another
specific embodiment, said selecting additionally comprises selecting a
population of placental cells that produces one or more embryoid-like
bodies when the population is cultured under conditions that allow the
formation of embryoid-like bodies.

[0069]The invention also provides an isolated stem cell that is
CD200+ and OCT-4+. In a specific embodiment, the stem cell is
CD73+ and CD105+. In a specific embodiment, the stem cell is a
placental stem cell. In another specific embodiment, said stem cell is
HLA-G+. In another specific embodiment, said stem cell is
CD34-, CD38- or CD45-. In another specific embodiment,
said stem cell is CD34-, CD38- and CD45-. In a more
specific embodiment, said stem cell is CD34, CD38, CD45, CD73+,
CD105+ and HLA-G-. In another specific embodiment, the stem
cell facilitates the production of one or more embryoid-like bodies by a
population of placental cells that comprises the stem cell, when the
population is cultured under conditions that allow the formation of
embryoid-like bodies. In another specific embodiment, said placental stem
cell is isolated away from placental cells that are not stem cells. In
another specific embodiment, said placental stem cell is isolated away
from placental stem cells that do not display these markers.

[0070]In another embodiment, the invention also provides a method of
selecting a placental stem cell from a plurality of placental cells,
comprising selecting a CD200+ and OCT-4+ placental cell,
whereby said cell is a placental stem cell. In a specific embodiment,
said selecting comprises selecting a placental cell that is also
HLA-G+. In another specific embodiment, said selecting comprises
selecting a placental cell that is also CD34-, CD38- or
CD45-. In another specific embodiment, said selecting comprises
selecting a placental cell that is also CD34-, CD38- and
CD45-. In another specific embodiment, said selecting comprises
selecting a placental cell that is also CD34-, CD38-,
CD45-, CD73+, CD105- and HLA-G+. In another specific
embodiment, said selecting comprises selecting a placental stem cell that
also facilitates the production of one or more embryoid-like bodies by a
population of placental cells that comprises the stem cell, when the
population is cultured under conditions that allow the formation of
embryoid-like bodies.

[0071]The invention also provides an isolated population of cells
comprising, e.g., that is enriched for, CD200+, OCT-4+ stem
cells. In various embodiments, at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, or at least
about 60% of said cells are CD200+, OCT-4+ stem cells. In
another embodiment, at least about 70% of said cells are said
CD200+, OCT-4+ stem cells. In another embodiment, at least
about 90%, 95%, or 99% of said cells are said CD200+, OCT-4+
stem cells. In a specific embodiment of the isolated populations, said
stem cells are CD73+ and CD105+. In another specific
embodiment, said stem cells are HLA-G+. In another specific
embodiment, said stem cells are CD34-, CD38- and CD45-. In
a more specific embodiment, said stem cells are CD34, CD38, CD45,
CD73+, CD105+ and HLA-G+. In another specific embodiment,
population produces one or more embryoid-like bodies when cultured under
conditions that allow the formation of embryoid-like bodies. In another
specific embodiment, said population of placental stem cells is isolated
away from placental cells that are not stem cells. In another specific
embodiment, said population of placental stem cells is isolated away from
placental stem cells that do not display these characteristics.

[0072]In another embodiment, the invention also provides a method of
selecting a placental stem cell population from a plurality of placental
cells, comprising selecting a population of placental cells wherein at
least about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50% at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or at least about 95% of said cells are
CD200+, OCT-4+ stem cells. In a specific embodiment, said
selecting comprises selecting stem cells that are also CD73+ and
CD105+. In another specific embodiment, said selecting comprises
selecting stem cells that are also HLA-G+. In another specific
embodiment, said selecting comprises selecting stem cells that are also
CD34-, CD38- and CD45-. In another specific embodiment,
said stem cells are also CD34-, CD38-, CD45-, CD73-,
CD105+ and HLA-G+.

[0073]The invention further provides an isolated stem cell that is
CD73+, CD105+ and HLA-G+. In a specific embodiment, the
stem cell is a placental stem cell. In another specific embodiment, said
stem cell is CD34-, CD38- or CD45-. In another specific
embodiment, said stem cell is CD34-, CD38- and CD45-. In
another specific embodiment, said stem cell is OCT-4+. In another
specific embodiment, said stem cell is CD200+. In a more specific
embodiment, said stem cell is CD34-, CD38-, CD45-,
OCT-4+ and CD200+. In another specific embodiment, said stem
cell facilitates the formation of embryoid-like bodies in a population of
placental cells comprising said stem cell, when the population is
cultured under conditions that allow the formation of embryoid-like
bodies. In another specific embodiment, said placental stem cell is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said placental stem cell is isolated away from
placental stem cells that do not display these characteristics.

[0074]In another embodiment, the invention also provides a method of
selecting a placental stem cell from a plurality of placental cells,
comprising selecting a CD73+, CD105+ and HLA-G+ placental
cell, whereby said cell is a placental stem cell. In a specific
embodiment, said selecting comprises selecting a placental cell that is
also CD34-, CD38- or CD45-. In another specific
embodiment, said selecting comprises selecting a placental cell that is
also CD34-, CD38- and CD45-. In another specific
embodiment, said selecting comprises selecting a placental cell that is
also OCT-4-. In another specific embodiment, said selecting
comprises selecting a placental cell that is also CD200+. In another
specific embodiment, said selecting comprises selecting a placental cell
that is also CD34-, CD38-, CD45-, OCT-4+ and
CD200+. In another specific embodiment, said selecting comprises
selecting a placental cell that also facilitates the formation of one or
more embryoid-like bodies in a population of placental cells that
comprises said stem cell, when said population is culture under
conditions that allow the formation of embryoid-like bodies.

[0075]The invention also provides an isolated population of cells
comprising, e.g., that is enriched for, CD73-, CD105+ and
HLA-G+ stem cells. In a specific embodiment, said stem cells are
placental stem cells. In various embodiments, at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least about
50%, or at least about 60% of said cells are CD73-, CD105+ and
HLA-G+ stem cells. In another embodiment, at least about 70% of said
cells are CD73+, CD105+ and HLA-G+. In another embodiment,
at least about 90%, 95% or 99% of said cells are CD73+, CD105+
and HLA-G+ stem cells. In a specific embodiment of the above
populations, said stem cells are CD34-, CD38- or CD45-. In
another specific embodiment, said stem cells are CD34-, CD38and CD45-. In another specific embodiment, said stem cells are
OCT-4+. In another specific embodiment, said stem cells are
CD200+. In a more specific embodiment, said stem cells are
CD34-, CD38-, CD45-, OCT-4+ and CD200+. In
another specific embodiment, said population of placental stem cells is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said population of placental stem cells is isolated
away from placental stem cells that do not display these characteristics.

[0076]In another embodiment, the invention also provides a method of
selecting a placental stem cell population from a plurality of placental
cells, comprising selecting a population of placental cells wherein a
majority of said cells are CD73+, CD105 and HLA-G+. In a
specific embodiment, said majority of cells are also CD34-,
CD38- and/or CD45-. In another specific embodiment, said
majority of cells are also CD200+. In another specific embodiment,
said majority of cells are also CD34-, CD38-, CD45-,
OCT-4+ and CD200+.

[0077]In another embodiment, the invention provides an isolated stem cell
that is CD73+ and CD105+ and which facilitates the formation of
one or more embryoid-like bodies in a population of isolated placental
cells comprising said stem cell when said population is cultured under
conditions that allow formation of embryoid-like bodies. In a specific
embodiment, said stem cell is CD34-, CD38- or CD45-. In
another specific embodiment, said stem cell is CD34-, CD38- and
CD45-. In another specific embodiment, said stem cell is OCT4+.
In a more specific embodiment, said stem cell is OCT4+, CD34, CD38
and CD45. In another specific embodiment, said placental stem cell is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said placental stem cell is isolated away from
placental stem cells that do not display these characteristics.

[0078]The invention further provides a population of isolated placental
cells comprising, e.g., that is enriched for, CD73+, CD105+
stem cells, wherein said population forms one or more embryoid-like
bodies under conditions that allow formation of embryoid-like bodies. In
various embodiments, at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50% at least about 60%, at
least about 70%, at least about 80%, at least about 90%, or at least
about 95% of said isolated placental cells are CD73+, CD105+
stem cells. In a specific embodiment of the above populations, said stem
cells are CD34-, CD38- or CD45-. In another specific
embodiment, said stem cells are CD34-, CD38- and CD45-. In
another specific embodiment, said stem cells are OCT-4+. In a more
specific embodiment, said stem cells are OCT-4+, CD34-,
CD38- and CD45-. In other specific embodiments, said population
has been expanded, for example, has been passaged at least once, at least
three times, at least five times, at least 10 times, at least 15 times,
or at least 20 times. In another specific embodiment, said population of
placental stem cells is isolated away from placental cells that are not
stem cells. In another specific embodiment, said population of placental
stem cells is isolated away from placental stem cells that do not display
these characteristics.

[0079]The invention further provides an isolated stem cell that is
OCT-4+ and which facilitates formation of one or more embryoid-like
bodies in a population of isolated placental cells comprising said stem
cell when cultured under conditions that allow formation of embryoid-like
bodies. In a specific embodiment, said stem cell is CD73+ and
CD105+. In another specific embodiment, said stem cell is
CD34-, CD38-, or CD45-. In another specific embodiment,
said stem cell is CD200+. In a more specific embodiment, said stem
cell is CD73+, CD105-, CD200+, CD34-, CD38-, and
CD45-. In another specific embodiment, said placental stem cell is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said placental stem cell is isolated away from
placental stem cells that do not display these characteristics.

[0080]The invention also provides a population of isolated cells
comprising, e.g., that is enriched for, OCT-4+ stem cells, wherein
said population forms one or more embryoid-like bodies when cultured
under conditions that allow the formation of embryoid-like bodies. In
various embodiments, at least 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50% at least about 60%, at least
about 70%, at least about 80%, at least about 90%, or at least about 95%
of said isolated placental cells are OCT4+ stem cells. In a specific
embodiment of the above populations, said stem cells are CD73+ and
CD105+. In another specific embodiment, said stem cells are
CD34-, CD38-, or CD45-. In another specific embodiment,
said stem cells are CD200+. In a more specific embodiment, said stem
cells are CD73-, CD105+, CD200+, CD34, CD38, and CD45. In
another specific embodiment, said population has been expanded, for
example, passaged at least once, at least three times, at least five
times, at least 10 times, at least 15 times, or at least 20 times. In
another specific embodiment, said population of placental stem cells is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said population of placental stem cells is isolated
away from placental stem cells that do not display these characteristics.

[0081]In another embodiment, the invention also provides an isolated
placental stem cell that is CD10+, CD34-, CD105+, and
CD200+. The invention further provides an isolated population of
placental stem cells, wherein at least about 70%, at least about 80%, at
least about 90%, at least about 95% or at least about 99% of said
placental stem cells are CD10+, CD34-, CD105+,
CD200+. In a specific embodiment of the above embodiments, said stem
cells are additionally CD90+ and CD45-. In a specific
embodiment, said stem cell or population of placental stem cells is
isolated away from placental cells that are not stem cells. In another
specific embodiment, said stem cell or population of placental stem cells
is isolated away from placental stem cells that do not display these
characteristics. In another specific embodiment, said isolated placental
stem cell is non-maternal in origin. In another specific embodiment, at
least about 90%, at least about 95%, or at least about 99% of said cells
in said isolated population of placental stem cells, are non-maternal in
origin.

[0082]In another embodiment, the invention provides an isolated placental
stem cell that is HLA-A,B,C-, CD45-, CD133- and
CD34-. The invention further provides an isolated population of
placental stem cells, wherein at least about 70%, at least about 80%, at
least about 90%, at least about 95% or at least about 99% of said
placental stem cells are HLA-A,B,C-, CD45-, CD133- and
CD34-. In a specific embodiment, said stem cell or population of
placental stem cells is isolated away from placental cells that are not
stem cells. In another specific embodiment, said population of placental
stem cells is isolated away from placental stem cells that do not display
these characteristics. In another specific embodiment, said isolated
placental stem cell is non-maternal in origin. In another specific
embodiment, at least about 90%, at least about 95%, or at least about 99%
of said cells in said isolated population of placental stem cells, are
non-maternal in origin. In another embodiment, the invention provides a
method of obtaining a placental stem cell that is HLA-A,B,C-,
CD45-, CD133- and CD34- comprising isolating said cell
from placental perfusate.

[0083]In another embodiment, the invention provides an isolated placental
stem cell that is CD10+, CD13+, CD33+, CD45, CD117 and
CD133. The invention further provides an isolated population of placental
stem cells, wherein at least about 70%, at least about 80%, at least
about 90%, at least about 95% or at least about 99% of said placental
stem cells are CD10+, CD13+, CD33+, CD45-,
CD117- and CD133-. In a specific embodiment, said stem cell or
population of placental stem cells is isolated away from placental cells
that are not stem cells. In another specific embodiment, said isolated
placental stem cell is non-maternal in origin. In another specific
embodiment, at least about 90%, at least about 95%, or at least about 99%
of said cells in said isolated population of placental stem cells, are
non-maternal in origin. In another specific embodiment, said stem cell or
population of placental stem cells is isolated away from placental stem
cells that do not display these characteristics. In another embodiment,
the invention provides a method of obtaining a placental stem cell that
is CD10+, CD13+, CD33+, CD45-, CD117- and
CD133- comprising isolating said cell from placental perfusate.

[0084]In another embodiment, the invention provides an isolated placental
stem cell that is CD10-, CD33-, CD44+, CD45-, and
CD117-. The invention further provides an isolated population of
placental stem cells, wherein at least about 70%, at least about 80%, at
least about 90%, at least about 95% or at least about 99% of said
placental stem cells are CD10-, CD33-, CD44+, CD45-,
and CD117-. In a specific embodiment, said stem cell or population
of placental stem cells is isolated away from placental cells that are
not stem cells. In another specific embodiment, said isolated placental
stem cell is non-maternal in origin. In another specific embodiment, at
least about 90%, at least about 95%, or at least 99% of said cells in
said isolated population of placental stem cells, are non-maternal in
origin. In another specific embodiment, said stem cell or population of
placental stem cells is isolated away from placental stem cells that do
not display these characteristics. In another embodiment, the invention
provides a method of obtaining a placental stem cell that is CD10-,
CD33-, CD44+, CD45-, CD117- comprising isolating said
cell from placental perfusate.

[0085]In another embodiment, the invention provides an isolated placental
stem cell that is CD10-, CD13-, CD33-, CD45-, and
CD117-. The invention further provides an isolated population of
placental stem cells, wherein at least about 70%, at least about 80%, at
least about 90%, at least about 95% or at least about 99% of said
placental stem cells are CD10-, CD13-, CD33-, CD45-,
and CD117-. In a specific embodiment, said stem cell or population
of placental stem cells is isolated away from placental cells that are
not stem cells. In another specific embodiment, said isolated placental
stem cell is non-maternal in origin. In another specific embodiment, at
least about 90%, at least about 95%, or at least 99% of said cells in
said isolated population of placental stem cells, are non-maternal in
origin. In another specific embodiment, said stem cell or population of
placental stem cells is isolated away from placental stem cells that do
not display these characteristics. In another embodiment, the invention
provides a method of obtaining a placental stem cell that is CD10-,
CD13-, CD33-, CD45-, and CD117+ comprising isolating
said cell from placental perfusate.

[0086]In another embodiment, the invention provides an isolated placental
stem cell that is HLA A,B,C-, CD45-, CD34-, CD133-,
positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G,
and/or negative for CD117. The invention further provides an isolated
population of placental stem cells, wherein said stem cells are HLA
A,B,C-, CD45-, CD34-, CD133-, and at least about 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98% or about 99% of the stem cells in the population are positive
for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or
negative for CD117. In a specific embodiment, said stem cell or
population of placental stem cells is isolated away from placental cells
that are not stem cells. In another specific embodiment, said isolated
placental stem cell is non-maternal in origin. In another specific
embodiment, at least about 90%, at least about 95%, or at least about
99%, of said cells in said isolated population of placental stem cells,
are non-maternal in origin. In another specific embodiment, said stem
cell or population of placental stem cells is isolated away from
placental stem cells that do not display these characteristics. In
another embodiment, the invention provides a method of obtaining a
placental stem cell that is HLA A,B,C-, CD45-, CD34-,
CD133- and positive for CD10, CD13, CD38, CD44, CD90, CD105, C200
and/or HLA-G, and/or negative for CD117, comprising isolating said cell
from placental perfusate.

[0087]In another embodiment, the invention provides a placental stem cell
that is CD200+ and CD10+, as determined by antibody binding,
and CD117-, as determined by both antibody binding and RT-PCR. In
another embodiment, the invention provides a placental stem cell that is
CD10+, CD29-, CD54+, CD200+, HLA-G+, HLA class
I- andβ-2-microglobul another embodiment, the invention
provides placental stem cells, wherein the expression of at least one
marker is at least two-fold higher than for a mesenchymal stem cell
(e.g., a bone marrow-derived mesenchymal stem cell). In another specific
embodiment, said isolated placental stem cell is non-maternal in origin.
In another specific embodiment, at least about 90%, at least about 95%,
or at least 99%, of said cells in said isolated population of placental
stem cells, are non-maternal in origin.

[0088]In another embodiment, the invention provides an isolated population
of placental stem cells, wherein a plurality of said placental stem cells
are positive for aldehyde dehydrogenase (ALDH), as assessed by an
aldehyde dehydrogenase activity assay. Such assays are known in the art
(see, e.g., Bostian and Betts, Biochem. J., 173, 787, (1978)). In a
specific embodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc.,
Ashland, Oreg.) as a marker of aldehyde dehydrogenase activity. In a
specific embodiment, said plurality is between about 3% and about 25% of
cells in said population of cells. In another embodiment, the invention
provides a population of umbilical cord stem cells, wherein a plurality
of said umbilical cord stem cells are positive for aldehyde
dehydrogenase, as assessed by an aldehyde dehydrogenase activity assay
that uses ALDEFLUOR® as an indicator of aldehyde dehydrogenase
activity. In a specific embodiment, said plurality is between about 3%
and about 25% of cells in said population of cells. In another
embodiment, said population of placental stem cells or umbilical cord
stem cells shows at least three-fold, or at least five-fold, higher ALDH
activity than a population of bone marrow-derived mesenchymal stem cells
having the same number of cells and cultured under the same conditions.

[0090]In a specific embodiment of any of the above placental cells or cell
populations, the karyotype of the cells, or at least about 95% or about
99% of the cells in said population, is normal. In another specific
embodiment of any of the above placental cells or cell populations, the
cells, or cells in the population of cells, are non-maternal in origin.

[0091]Isolated placental stem cells, or isolated populations of placental
stem cells, bearing any of the above combinations of markers, can be
combined in any ratio. The invention also provides for the isolation of,
or enrichment for, any two or more of the above placental stem cell
populations to form a placental stem cell population. For example, the
invention provides an isolated population of placental stem cells
comprising a first population of placental stem cells defined by one of
the marker combinations described above and a second population of
placental stem cells defined by another of the marker combinations
described above, wherein said first and second populations are combined
in a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70,
40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or
about 99:1. In like fashion, any three, four, five or more of the
above-described placental stem cells or placental stem cell populations
can be combined.

[0092]The invention further provides placental stem cells that are
obtained by disruption of placental tissue, with or without enzymatic
digestion, followed by culture (see Section 5.2.3) or perfusion (see
Section 5.2.4). For example, the invention provides an isolated
population of placental stem cells that is produced according to a method
comprising perfusing a mammalian placenta that has been drained of cord
blood and perfused to remove residual blood; perfusing said placenta with
a perfusion solution; and collecting said perfusion solution, wherein
said perfusion solution after perfusion comprises a population of
placental cells that comprises placental stem cells; and isolating a
plurality of said placental stem cells from said population of cells. In
a specific embodiment, the perfusion solution is passed through both the
umbilical vein and umbilical arteries and collected after it exudes from
the placenta. Populations of placental stem cells produced by this method
typically comprise a mixture of fetal and maternal cells. In another
specific embodiment, the perfusion solution is passed through the
umbilical vein and collected from the umbilical arteries, or passed
through the umbilical arteries and collected from the umbilical vein.
Populations of placental stem cells produced by this method typically are
substantially exclusively fetal in origin; that is, e.g., greater than
90%, 95%, 99%, or 99.5% of the placental stem cells in the population are
fetal in origin.

[0093]In various embodiments, the placental stem cells, contained within a
population of cells obtained from perfusion of a placenta, are at least
50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said population of
placental cells. In another specific embodiment, the placental stem cells
collected by perfusion comprise fetal and maternal cells. In another
specific embodiment, the placental stem cells collected by perfusion are
at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% fetal cells.

[0094]In another specific embodiment, the invention provides a composition
comprising a population of isolated placental stem cells collected by
perfusion, wherein said composition comprises at least a portion of the
perfusion solution used to collect the placental stem cells.

[0095]The invention further provides an isolated population of the
placental stem cells described herein that is produced according to a
method comprising digesting placental tissue with a tissue-disrupting
enzyme to obtain a population of placental cells comprising placental
stem cells, and isolating a plurality of placental stem cells from the
remainder of said placental cells. The whole, or any part of, the
placenta can be digested to obtain placental stem cells. In specific
embodiments, for example, said placental tissue is a whole placenta, an
amniotic membrane, chorion, a combination of amnion and chorion, or a
combination of any of the foregoing. In other specific embodiment, the
tissue-disrupting enzyme is trypsin or collagenase. In various
embodiments, the placental stem cells, contained within a population of
cells obtained from digesting a placenta, are at least 50%, 60%, 70%,
80%, 90%, 95%, 99% or at least 99.5% of said population of placental
cells.

[0096]Gene profiling confirms that isolated placental stem cells, and
populations of isolated placental stem cells, are distinguishable from
other cells, e.g., mesenchymal stem cells, e.g., bone marrow-derived stem
cells. The placental stem cells described herein, can be distinguished
from mesenchymal stem cells on the basis of the expression of one or more
genes, the expression of which is specific to placental stem cells or
umbilical cord stem cells in comparison to bone marrow-derived
mesenchymal stem cells. In particular, placental stem cells can be
distinguished from mesenchymal stem cells on the basis of the expression
of one or more gene, the expression of which is significantly higher
(that is, at least twofold higher) in placental stem cells than in
mesenchymal stem cells, wherein the one or more gene is(are) ACTG2,
ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2,
CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B,
HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP,
MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6,
ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, ZC3H12A, or a combination of any
of the foregoing, wherein the expression of these genes is higher in
placental stem cells or umbilical cord stem cells than in bone
marrow-derived stem cells, when the stem cells are grown under equivalent
conditions. In a specific embodiment, the placental stem cell-specific or
umbilical cord stem cell-specific gene is CD200.

[0097]The level of expression of these genes can be used to confirm the
identity of a population of placental cells, to identify a population of
cells as comprising at least a plurality of placental stem cells, or the
like. The population of placental stem cells, the identity of which is
confirmed, can be clonal, e.g., a population of placental stem cells
expanded form a single placental stem cell, or a mixed population of stem
cells, e.g., a population of cells comprising solely placental stem cells
that are expanded from multiple placental stem cells, or a population of
cells comprising placental stem cells and at least one other type of
cell.

[0098]The level of expression of these genes can be used to select
populations of placental stem cells. For example, a population of cells,
e.g., clonally-expanded cells, is selected if the expression of one or
more of these genes is significantly higher in a sample from the
population of cells than in an equivalent population of mesenchymal stem
cells. Such selecting can be of a population from a plurality of
placental stem cells populations, from a plurality of cell populations,
the identity of which is not known, etc.

[0099]Placental stem cells can be selected on the basis of the level of
expression of one or more such genes as compared to the level of
expression in said one or more genes in a mesenchymal stem cell control.
In one embodiment, the level of expression of said one or more genes in a
sample comprising an equivalent number of mesenchymal stem cells is used
as a control. In another embodiment, the control, for placental stem
cells tested under certain conditions, is a numeric value representing
the level of expression of said one or more genes in mesenchymal stem
cells under said conditions.

[0103]The growth of the placental stem cells described herein, as for any
mammalian cell, depends in part upon the particular medium selected for
growth. Under optimum conditions, placental stem cells typically double
in number in 3-5 days. During culture, the placental stem cells of the
invention adhere to a substrate in culture, e.g. the surface of a tissue
culture container (e.g., tissue culture dish plastic, fibronectin-coated
plastic, and the like) and form a monolayer.

[0104]Populations of isolated placental cells that comprise the placental
stem cells of the invention, when cultured under appropriate conditions,
form embryoid-like bodies, that is, three-dimensional clusters of cells
grow atop the adherent stem cell layer. Cells within the embryoid-like
bodies express markers associated with very early stem cells, e.g.,
OCT-4, Nanog, SSEA3 and SSEA4. Cells within the embryoid-like bodies are
typically not adherent to the culture substrate, as are the placental
stem cells described herein, but remain attached to the adherent cells
during culture. Embryoid-like body cells are dependent upon the adherent
placental stem cells for viability, as embryoid-like bodies do not form
in the absence of the adherent stem cells. The adherent placental stem
cells thus facilitate the growth of one or more embryoid-like bodies in a
population of placental cells that comprise the adherent placental stem
cells. Without wishing to be bound by theory, the cells of the
embryoid-like bodies are thought to grow on the adherent placental stem
cells much as embryonic stem cells grow on a feeder layer of cells.
Mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells,
do not develop embryoid-like bodies in culture.

[0108]The stem cell collection composition can comprise one or more
components that tend to preserve placental stem cells, that is, prevent
the placental stem cells from dying, or delay the death of the placental
stem cells, reduce the number of placental stem cells in a population of
cells that die, or the like, from the time of collection to the time of
culturing. Such components can be, e.g., an apoptosis inhibitor (e.g., a
caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium
sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),
adrenocorticotropin, corticotropin-releasing hormone, sodium
nitroprusside, hydralazine, adenosine triphosphate, adenosine,
indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);
a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,
pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor;
and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,
perfluorodecyl bromide, etc.).

[0109]The stem cell collection composition can comprise one or more
tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a
neutral protease, an RNase, or a DNase, or the like. Such enzymes
include, but are not limited to, collagenases (e.g., collagenase I, II,
III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,
thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

[0110]The stem cell collection composition can comprise a bacteriocidally
or bacteriostatically effective amount of an antibiotic. In certain
non-limiting embodiments, the antibiotic is a macrolide (e.g.,
tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,
cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an
erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,
ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin,
etc. In a particular embodiment, the antibiotic is active against Gram(+)
and/or Gram(-) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus
aureus, and the like.

[0111]The stem cell collection composition can also comprise one or more
of the following compounds: adenosine (about 1 mM to about 50 mM);
D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to
about 50 mM); a macromolecule of molecular weight greater than 20,000
daltons, in one embodiment, present in an amount sufficient to maintain
endothelial integrity and cellular viability (e.g., a synthetic or
naturally occurring colloid, a polysaccharide such as dextran or a
polyethylene glycol present at about 25 g/l to about 100 g/l, or about 40
g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole,
butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at
about 25 μM to about 100 μM); a reducing agent (e.g.,
N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent that
prevents calcium entry into cells (e.g., verapamil present at about 2
μM to about 25 μM); nitroglycerin (e.g., about 0.05 g/L to about
0.2 g/L); an anticoagulant, in one embodiment, present in an amount
sufficient to help prevent clotting of residual blood (e.g., heparin or
hirudin present at a concentration of about 1000 units/l to about 100,000
units/l); or an amiloride containing compound (e.g., amiloride, ethyl
isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or
isobutyl amiloride present at about 1.0 μM to about 5 μM).

[0112]5.2.2 Collection and Handling of Placenta

[0113]Generally, a human placenta is recovered shortly after its expulsion
after birth. In a preferred embodiment, the placenta is recovered from a
patient after informed consent and after a complete medical history of
the patient is taken and is associated with the placenta. Preferably, the
medical history continues after delivery. Such a medical history can be
used to coordinate subsequent use of the placenta or the stem cells
harvested therefrom. For example, human placental stem cells can be used,
in light of the medical history, for personalized medicine for the infant
associated with the placenta, or for parents, siblings or other relatives
of the infant.

[0114]Prior to recovery of placental stem cells, the umbilical cord blood
and placental blood are removed. In certain embodiments, after delivery,
the cord blood in the placenta is recovered. The placenta can be
subjected to a conventional cord blood recovery process. Typically a
needle or cannula is used, with the aid of gravity, to exsanguinate the
placenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al.,
U.S. Pat. No. 5,415,665). The needle or cannula is usually placed in the
umbilical vein and the placenta can be gently massaged to aid in draining
cord blood from the placenta. Such cord blood recovery may be performed
commercially, e.g., LifeBank USA, Cedar Knolls, N.J., ViaCord, Cord Blood
Registry and Cryocell. Preferably, the placenta is gravity drained
without further manipulation so as to minimize tissue disruption during
cord blood recovery.

[0115]Typically, a placenta is transported from the delivery or birthing
room to another location, e.g., a laboratory, for recovery of cord blood
and collection of stem cells by, e.g., perfusion or tissue dissociation.
The placenta is preferably transported in a sterile, thermally insulated
transport device (maintaining the temperature of the placenta between
20-28° C.), for example, by placing the placenta, with clamped
proximal umbilical cord, in a sterile zip-lock plastic bag, which is then
placed in an insulated container. In another embodiment, the placenta is
transported in a cord blood collection kit substantially as described in
pending U.S. Pat. No. 7,147,626. Preferably, the placenta is delivered to
the laboratory four to twenty-four hours following delivery. In certain
embodiments, the proximal umbilical cord is clamped, preferably within
4-5 cm (centimeter) of the insertion into the placental disc prior to
cord blood recovery. In other embodiments, the proximal umbilical cord is
clamped after cord blood recovery but prior to further processing of the
placenta.

[0116]The placenta, prior to stem cell collection, can be stored under
sterile conditions and at either room temperature or at a temperature of
5 to 25° C. (centigrade). The placenta may be stored for a period
of for a period of four to twenty-four hours, up to forty-eight hours, or
longer than forty eight hours, prior to perfusing the placenta to remove
any residual cord blood. In one embodiment, the placenta is harvested
from between about zero hours to about two hours post-expulsion. The
placenta is preferably stored in an anticoagulant solution at a
temperature of 5 to 25° C. (centigrade). Suitable anticoagulant
solutions are well known in the art. For example, a solution of heparin
or warfarin sodium can be used. In a preferred embodiment, the
anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in
1:1000 solution). The exsanguinated placenta is preferably stored for no
more than 36 hours before placental stem cells are collected.

[0117]The mammalian placenta or a part thereof, once collected and
prepared generally as above, can be treated in any art-known manner,
e.g., can be perfused or disrupted, e.g., digested with one or more
tissue-disrupting enzymes, to obtain stem cells.

[0119]In one embodiment, stem cells are collected from a mammalian
placenta by physical disruption of part of all of the organ. For example,
the placenta, or a portion thereof, may be, e.g., crushed, sheared,
minced, diced, chopped, macerated or the like. The tissue can then be
cultured to obtain a population of stem cells. Typically, the placental
tissue is disrupted using, e.g., in, a stem cell collection composition
(see Section 5.2.1 and below).

[0120]The placenta can be dissected into components prior to physical
disruption and/or enzymatic digestion and stem cell recovery. Placental
stem cells can be obtained from all or a portion of the amniotic
membrane, chorion, umbilical cord, placental cotyledons, or any
combination thereof, including from a whole placenta. Preferably,
placental stem cells are obtained from placental tissue comprising amnion
and chorion. Typically, placental stem cells can be obtained by
disruption of a small block of placental tissue, e.g., a block of
placental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about
1000 cubic millimeters in volume. Any method of physical disruption can
be used, provided that the method of disruption leaves a plurality, more
preferably a majority, and more preferably at least 60%, 70%, 80%, 90%,
95%, 98%, or 99% of the cells in said organ viable, as determined by,
e.g., trypan blue exclusion.

[0121]Stem cells can generally be collected from a placenta, or portion
thereof, at any time within about the first three days post-expulsion,
but preferably between about 8 hours and about 18 hours post-expulsion.

[0123]In another specific embodiment, stem cells are collected by physical
disruption of placental tissue, wherein the physical disruption includes
enzymatic digestion, which can be accomplished by use of one or more
tissue-digesting enzymes. The placenta, or a portion thereof, may also be
physically disrupted and digested with one or more enzymes, and the
resulting material then immersed in, or mixed into, a stem cell
collection composition.

[0124]A preferred stem cell collection composition comprises one or more
tissue-disruptive enzyme(s). Enzymatic digestion preferably uses a
combination of enzymes, e.g., a combination of a matrix metalloprotease
and a neutral protease, for example, a combination of collagenase and
dispase. In one embodiment, enzymatic digestion of placental tissue uses
a combination of a matrix metalloprotease, a neutral protease, and a
mucolytic enzyme for digestion of hyaluronic acid, such as a combination
of collagenase, dispase, and hyaluronidase or a combination of LIBERASE
(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other
enzymes that can be used to disrupt placenta tissue include papain,
deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, or
elastase. Serine proteases may be inhibited by alpha 2 microglobulin in
serum and therefore the medium used for digestion is usually serum-free.
EDTA and DNase are commonly used in enzyme digestion procedures to
increase the efficiency of cell recovery. The digestate is preferably
diluted so as to avoid trapping stem cells within the viscous digest.

[0125]Any combination of tissue digestion enzymes can be used. Typical
concentrations for tissue digestion enzymes include, e.g., 50-200 U/mL
for collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100
U/mL for elastase. Proteases can be used in combination, that is, two or
more proteases in the same digestion reaction, or can be used
sequentially in order to liberate placental stem cells. For example, in
one embodiment, a placenta, or part thereof, is digested first with an
appropriate amount of collagenase I at about 1 to about 2 mg/ml for,
e.g., 30 minutes, followed by digestion with trypsin, at a concentration
of about 0.25%, for, e.g., 10 minutes, at 37° C. Serine proteases
are preferably used consecutively following use of other enzymes.

[0126]In another embodiment, the tissue can further be disrupted by the
addition of a chelator, e.g., ethylene glycol bis(2-aminoethyl
ether)-N,N,N'N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic
acid (EDTA) to the stem cell collection composition comprising the stem
cells, or to a solution in which the tissue is disrupted and/or digested
prior to isolation of the stem cells with the stem cell collection
composition.

[0127]In one embodiment, a digestion can proceed as follows. Approximately
a gram of placental tissue is obtained and minced. The tissue is digested
in 10 mL of a solution comprising about 1 mg/mL collagenase 1A and about
0.25% trypsin at 37° C. in a shaker at about 100 RPM. The
digestate is washed three times with culture medium, and the washed cells
are seeded into 2 T-75 flasks. The cells are then isolated by
differential adherence, and characterized for, e.g., viability, cell
surface markers, differentiation, and the like.

[0128]It will be appreciated that where an entire placenta, or portion of
a placenta comprising both fetal and maternal cells (for example, where
the portion of the placenta comprises the chorion or cotyledons), the
placental stem cells collected will comprise a mix of placental stem
cells derived from both fetal and maternal sources. Where a portion of
the placenta that comprises no, or a negligible number of, maternal cells
(for example, amnion), the placental stem cells collected will comprise
almost exclusively fetal placental stem cells.

[0129]Stem cells can be isolated from disrupted tissue by differential
trypsinization (see Section 5.2.5, below) followed by culture in one or
more new culture containers in fresh proliferation medium, optionally
followed by a second differential trypsinization step.

[0130]5.2.4 Placental Perfusion

[0131]Placental stem cells can also be obtained by perfusion of the
mammalian placenta. Methods of perfusing mammalian placenta to obtain
stem cells are disclosed, e.g., in Hariri, U.S. Application Publication
No. 2002/0123141, and in related U.S. Provisional Application No.
60/754,969, entitled "Improved Medium for Collecting Placental Stem Cells
and Preserving Organs," filed on Dec. 29, 2005.

[0132]Placental stem cells can be collected by perfusion, e.g., through
the placental vasculature, using, e.g., a stem cell collection
composition as a perfusion solution. In one embodiment, a mammalian
placenta is perfused by passage of perfusion solution through either or
both of the umbilical artery and umbilical vein. The flow of perfusion
solution through the placenta may be accomplished using, e.g., gravity
flow into the placenta. Preferably, the perfusion solution is forced
through the placenta using a pump, e.g., a peristaltic pump. The
umbilical vein can be, e.g., cannulated with a cannula, e.g., a
TEFLON® or plastic cannula, that is connected to a sterile connection
apparatus, such as sterile tubing. The sterile connection apparatus is
connected to a perfusion manifold.

[0133]In preparation for perfusion, the placenta is preferably oriented
(e.g., suspended) in such a manner that the umbilical artery and
umbilical vein are located at the highest point of the placenta. The
placenta can be perfused by passage of a perfusion fluid through the
placental vasculature and surrounding tissue. The placenta can also be
perfused by passage of a perfusion fluid into the umbilical vein and
collection from the umbilical arteries, or passage of a perfusion fluid
into the umbilical arteries and collection from the umbilical vein.

[0134]In one embodiment, for example, the umbilical artery and the
umbilical vein are connected simultaneously, e.g., to a pipette that is
connected via a flexible connector to a reservoir of the perfusion
solution. The perfusion solution is passed into the umbilical vein and
artery. The perfusion solution exudes from and/or passes through the
walls of the blood vessels into the surrounding tissues of the placenta,
and is collected in a suitable open vessel from the surface of the
placenta that was attached to the uterus of the mother during gestation.
The perfusion solution may also be introduced through the umbilical cord
opening and allowed to flow or percolate out of openings in the wall of
the placenta which interfaced with the maternal uterine wall. Placental
cells that are collected by this method, which can be referred to as a
"pan" method, are typically a mixture of fetal and maternal cells.

[0135]In another embodiment, the perfusion solution is passed through the
umbilical veins and collected from the umbilical artery, or is passed
through the umbilical artery and collected from the umbilical veins.
Placental cells collected by this method, which can be referred to as a
"closed circuit" method, are typically almost exclusively fetal.

[0136]It will be appreciated that perfusion using the pan method, that is,
whereby perfusate is collected after it has exuded from the maternal side
of the placenta, results in a mix of fetal and maternal cells. As a
result, the cells collected by this method comprise a mixed population of
placental stem cells of both fetal and maternal origin. In contrast,
perfusion solely through the placental vasculature in the closed circuit
method, whereby perfusion fluid is passed through one or two placental
vessels and is collected solely through the remaining vessel(s), results
in the collection of a population of placental stem cells almost
exclusively of fetal origin.

[0137]The closed circuit perfusion method can, in one embodiment, be
performed as follows. A post-partum placenta is obtained within about 48
hours after birth. The umbilical cord is clamped and cut above the clamp.
The umbilical cord can be discarded, or can processed to recover, e.g.,
umbilical cord stem cells, and/or to process the umbilical cord membrane
for the production of a biomaterial. The amniotic membrane can be
retained during perfusion, or can be separated from the chorion, e.g.,
using blunt dissection with the fingers. If the amniotic membrane is
separated from the chorion prior to perfusion, it can be, e.g.,
discarded, or processed, e.g., to obtain stem cells by enzymatic
digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g.,
the biomaterial described in U.S. Application Publication No.
2004/0048796. After cleaning the placenta of all visible blood clots and
residual blood, e.g., using sterile gauze, the umbilical cord vessels are
exposed, e.g., by partially cutting the umbilical cord membrane to expose
a cross-section of the cord. The vessels are identified, and opened,
e.g., by advancing a closed alligator clamp through the cut end of each
vessel. The apparatus, e.g., plastic tubing connected to a perfusion
device or peristaltic pump, is then inserted into each of the placental
arteries. The pump can be any pump suitable for the purpose, e.g., a
peristaltic pump. Plastic tubing, connected to a sterile collection
reservoir, e.g., a blood bag such as a 250 mL collection bag, is then
inserted into the placental vein. Alternatively, the tubing connected to
the pump is inserted into the placental vein, and tubes to a collection
reservoir(s) are inserted into one or both of the placental arteries. The
placenta is then perfused with a volume of perfusion solution, e.g.,
about 750 ml of perfusion solution. Cells in the perfusate are then
collected, e.g., by centrifugation.

[0138]In one embodiment, the proximal umbilical cord is clamped during
perfusion, and more preferably, is clamped within 4-5 cm (centimeter) of
the cord's insertion into the placental disc.

[0139]The first collection of perfusion fluid from a mammalian placenta
during the exsanguination process is generally colored with residual red
blood cells of the cord blood and/or placental blood. The perfusion fluid
becomes more colorless as perfusion proceeds and the residual cord blood
cells are washed out of the placenta. Generally from 30 to 100 ml
(milliliter) of perfusion fluid is adequate to initially exsanguinate the
placenta, but more or less perfusion fluid may be used depending on the
observed results.

[0140]The volume of perfusion liquid used to collect placental stem cells
may vary depending upon the number of stem cells to be collected, the
size of the placenta, the number of collections to be made from a single
placenta, etc. In various embodiments, the volume of perfusion liquid may
be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to
2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.
Typically, the placenta is perfused with 700-800 mL of perfusion liquid
following exsanguination.

[0141]The placenta can be perfused a plurality of times over the course of
several hours or several days. Where the placenta is to be perfused a
plurality of times, it may be maintained or cultured under aseptic
conditions in a container or other suitable vessel, and perfused with the
stem cell collection composition, or a standard perfusion solution (e.g.,
a normal saline solution such as phosphate buffered saline ("PBS")) with
or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin,
bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g.,
β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g.,
at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g.,
at 0.5 μg/ml). In one embodiment, an isolated placenta is maintained
or cultured for a period of time without collecting the perfusate, such
that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours,
or 2 or 3 or more days before perfusion and collection of perfusate. The
perfused placenta can be maintained for one or more additional time(s),
e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g.,
700-800 mL perfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or
more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a
preferred embodiment, perfusion of the placenta and collection of
perfusion solution, e.g., stem cell collection composition, is repeated
until the number of recovered nucleated cells falls below 100 cells/ml.
The perfusates at different time points can be further processed
individually to recover time-dependent populations of cells, e.g., stem
cells. Perfusates from different time points can also be pooled. In a
preferred embodiment, stem cells are collected at a time or times between
about 8 hours and about 18 hours post-expulsion.

[0142]Without wishing to be bound by any theory, after exsanguination and
a sufficient time of perfusion of the placenta, placental stem cells are
believed to migrate into the exsanguinated and perfused microcirculation
of the placenta where, according to the methods of the invention, they
are collected, preferably by washing into a collecting vessel by
perfusion. Perfusing the isolated placenta not only serves to remove
residual cord blood but also provide the placenta with the appropriate
nutrients, including oxygen. The placenta may be cultivated and perfused
with a similar solution which was used to remove the residual cord blood
cells, preferably, without the addition of anticoagulant agents.

[0143]Perfusion according to the methods of the invention results in the
collection of significantly more placental stem cells than the number
obtainable from a mammalian placenta not perfused with said solution, and
not otherwise treated to obtain stem cells (e.g., by tissue disruption,
e.g., enzymatic digestion). In this context, "significantly more" means
at least 10% more. Perfusion according to the methods of the invention
yields significantly more placental stem cells than, e.g., the number of
placental stem cells obtainable from culture medium in which a placenta,
or portion thereof, has been cultured.

[0144]Stem cells can be isolated from placenta by perfusion with a
solution comprising one or more proteases or other tissue-disruptive
enzymes. In a specific embodiment, a placenta or portion thereof (e.g.,
amniotic membrane, amnion and chorion, placental lobule or cotyledon,
umbilical cord, or combination of any of the foregoing) is brought to
25-37° C., and is incubated with one or more tissue-disruptive
enzymes in 200 mL of a culture medium for 30 minutes. Cells from the
perfusate are collected, brought to 4° C., and washed with a cold
inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM
beta-mercaptoethanol. The stem cells are washed after several minutes
with a cold (e.g., 4° C.) stem cell collection composition.

[0146]Stem cells from mammalian placenta, whether obtained by perfusion or
enyzmatic digestion, can initially be purified from (i.e., be isolated
from) other cells by Ficoll gradient centrifugation. Such centrifugation
can follow any standard protocol for centrifugation speed, etc. In one
embodiment, for example, cells collected from the placenta are recovered
from perfusate by centrifugation at 5000×g for 15 minutes at room
temperature, which separates cells from, e.g., contaminating debris and
platelets. In another embodiment, placental perfusate is concentrated to
about 200 ml, gently layered over Ficoll, and centrifuged at about
1100×g for 20 minutes at 22° C., and the low-density
interface layer of cells is collected for further processing.

[0148]As used herein, "isolating" placental stem cells means to remove at
least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells
with which the stem cells are normally associated in the intact mammalian
placenta. A stem cell from an organ is "isolated" when it is present in a
population of cells that comprises fewer than 50% of the cells with which
the stem cell is normally associated in the intact organ.

[0149]Placental cells obtained by perfusion or digestion can, for example,
be further, or initially, isolated by differential trypsinization using,
e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).
Differential trypsinization is possible because placental stem cells
typically detach from plastic surfaces within about five minutes whereas
other adherent populations typically require more than 20-30 minutes
incubation. The detached placental stem cells can be harvested following
trypsinization and trypsin neutralization, using, e.g., Trypsin
Neutralizing Solution (TNS, Cambrex). In one embodiment of isolation of
adherent cells, aliquots of, for example, about 5-10×106 cells
are placed in each of several T-75 flasks, preferably fibronectin-coated
T75 flasks. In such an embodiment, the cells can be cultured with
commercially available Mesenchymal Stem Cell Growth Medium (MSCGM)
(Cambrex), and placed in a tissue culture incubator (37° C., 5%
CO2). After 10 to 15 days, non-adherent cells are removed from the
flasks by washing with PBS. The PBS is then replaced by MSCGM. Flasks are
preferably examined daily for the presence of various adherent cell types
and in particular, for identification and expansion of clusters of
fibroblastoid cells.

[0150]The number and type of cells collected from a mammalian placenta can
be monitored, for example, by measuring changes in morphology and cell
surface markers using standard cell detection techniques such as flow
cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue
specific or cell-marker specific antibodies) fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), by examination of
the morphology of cells using light or confocal microscopy, and/or by
measuring changes in gene expression using techniques well known in the
art, such as PCR and gene expression profiling. These techniques can be
used, too, to identify cells that are positive for one or more particular
markers. For example, using antibodies to CD34, one can determine, using
the techniques above, whether a cell comprises a detectable amount of
CD34; if so, the cell is CD34+. Likewise, if a cell produces enough
OCT-4 RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA
than an adult cell, the cell is OCT-4+ Antibodies to cell surface
markers (e.g., CD markers such as CD34) and the sequence of stem
cell-specific genes, such as OCT-4, are well-known in the art.

[0151]Placental cells, particularly cells that have been isolated by
Ficoll separation, differential adherence, or a combination of both, may
be sorted using a fluorescence activated cell sorter (FACS). Fluorescence
activated cell sorting (FACS) is a well-known method for separating
particles, including cells, based on the fluorescent properties of the
particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation
of fluorescent moieties in the individual particles results in a small
electrical charge allowing electromagnetic separation of positive and
negative particles from a mixture. In one embodiment, cell surface
marker-specific antibodies or ligands are labeled with distinct
fluorescent labels. Cells are processed through the cell sorter, allowing
separation of cells based on their ability to bind to the antibodies
used. FACS sorted particles may be directly deposited into individual
wells of 96-well or 384-well plates to facilitate separation and cloning.

[0152]In one sorting scheme, stem cells from placenta are sorted on the
basis of expression of the markers CD34, CD38, CD44, CD45, CD73, CD105,
OCT-4 and/or HLA-G. This can be accomplished in connection with
procedures to select stem cells on the basis of their adherence
properties in culture. For example, an adherence selection stem can be
accomplished before or after sorting on the basis of marker expression.
In one embodiment, for example, cells are sorted first on the basis of
their expression of CD34; CD34- cells are retained, and cells that
are CD200+HLA-G+, are separated from all other CD34cells. In another embodiment, cells from placenta are based on their
expression of markers CD200 and/or HLA-G; for example, cells displaying
either of these markers are isolated for further use. Cells that express,
e.g., CD200 and/or HLA-G can, in a specific embodiment, be further sorted
based on their expression of CD73 and/or CD105, or epitopes recognized by
antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45.
For example, in one embodiment, placental cells are sorted by expression,
or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and
placental cells that are CD200+, HLA-G+, CD73+,
CD105-, CD34-, CD38- and CD45- are isolated from
other placental cells for further use.

[0154]Other antibody/label combinations that can be used include, but are
not limited to, CD45-PerCP (peridin chlorophyll protein); CD44-PE;
CD19-PE; CD10-F (fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD);
HLA-ABC-F; and the like.

[0155]Placental stem cells can be assayed for CD117 or CD133 using, for
example, phycoerythrin-Cy5 (PE Cy5) conjugated streptavidin and biotin
conjugated monoclonal antibodies against CD117 or CD133; however, using
this system, the cells can appear to be positive for CD117 or CD133,
respectively, because of a relatively high background.

[0156]Placental stem cells can be labeled with an antibody to a single
marker and detected and/sorted. Placental stem cells can also be
simultaneously labeled with multiple antibodies to different markers.

[0157]In another embodiment, magnetic beads can be used to separate cells.
The cells may be sorted using a magnetic activated cell sorting (MACS)
technique, a method for separating particles based on their ability to
bind magnetic beads (0.5-100 μm diameter). A variety of useful
modifications can be performed on the magnetic microspheres, including
covalent addition of antibody that specifically recognizes a particular
cell surface molecule or hapten. The beads are then mixed with the cells
to allow binding. Cells are then passed through a magnetic field to
separate out cells having the specific cell surface marker. In one
embodiment, these cells can then isolated and re-mixed with magnetic
beads coupled to an antibody against additional cell surface markers. The
cells are again passed through a magnetic field, isolating cells that
bound both the antibodies. Such cells can then be diluted into separate
dishes, such as microtiter dishes for clonal isolation.

[0158]Placental stem cells can also be characterized and/or sorted based
on cell morphology and growth characteristics. For example, placental
stem cells can be characterized as having, and/or selected on the basis
of, e.g., a fibroblastoid appearance in culture. Placental stem cells can
also be characterized as having, and/or be selected, on the basis of
their ability to form embryoid-like bodies. In one embodiment, for
example, placental cells that are fibroblastoid in shape, express CD73
and CD105, and produce one or more embryoid-like bodies in culture are
isolated from other placental cells. In another embodiment, OCT-4+
placental cells that produce one or more embryoid-like bodies in culture
are isolated from other placental cells.

[0159]In another embodiment, placental stem cells can be identified and
characterized by a colony forming unit assay. Colony forming unit assays
are commonly known in the art, such as MESEN CULT® medium (Stem Cell
Technologies, Inc., Vancouver British Columbia)

[0160]Placental stem cells can be assessed for viability, proliferation
potential, and longevity using standard techniques known in the art, such
as trypan blue exclusion assay, fluorescein diacetate uptake assay,
propidium iodide uptake assay (to assess viability); and thymidine uptake
assay, MTT cell proliferation assay (to assess proliferation). Longevity
may be determined by methods well known in the art, such as by
determining the maximum number of population doubling in an extended
culture.

[0161]Placental stem cells can also be separated from other placental
cells using other techniques known in the art, e.g., selective growth of
desired cells (positive selection), selective destruction of unwanted
cells (negative selection); separation based upon differential cell
agglutinability in the mixed population as, for example, with soybean
agglutinin; freeze-thaw procedures; filtration; conventional and zonal
centrifugation; centrifugal elutriation (counter-streaming
centrifugation); unit gravity separation; countercurrent distribution;
electrophoresis; and the like.

[0167]Placental stem cells can be cultured in standard tissue culture
conditions, e.g., in tissue culture dishes or multiwell plates. Placental
stem cells can also be cultured using a hanging drop method. In this
method, placental stem cells are suspended at about 1×104
cells per mL in about 5 mL of medium, and one or more drops of the medium
are placed on the inside of the lid of a tissue culture container, e.g.,
a 100 mL Petri dish. The drops can be, e.g., single drops, or multiple
drops from, e.g., a multichannel pipetter. The lid is carefully inverted
and placed on top of the bottom of the dish, which contains a volume of
liquid, e.g., sterile PBS sufficient to maintain the moisture content in
the dish atmosphere, and the stem cells are cultured.

[0168]In one embodiment, the placental stem cells are cultured in the
presence of a compound that acts to maintain an undifferentiated
phenotype in the placental stem cell. In a specific embodiment, the
compound is a substituted 3,4-dihydropyridimol[4,5-d]pyrimidine. In a
more specific embodiment, the compound is a compound having the following
chemical structure:

##STR00001##

The compound can be contacted with a placental stem cell, or population of
placental stem cells, at a concentration of, for example, between about 1
μM to about 10 μM.

[0169]5.3.2 Expansion and Proliferation of Placental Stem Cells

[0170]Once an isolated placental stem cell, or isolated population of stem
cells (e.g., a stem cell or population of stem cells separated from at
least 50% of the placental cells with which the stem cell or population
of stem cells is normally associated in vivo), the stem cell or
population of stem cells can be proliferated and expanded in vitro. For
example, a population of placental stem cells can be cultured in tissue
culture containers, e.g., dishes, flasks, multiwell plates, or the like,
for a sufficient time for the stem cells to proliferate to 70-90%
confluence, that is, until the stem cells and their progeny occupy 70-90%
of the culturing surface area of the tissue culture container.

[0171]Placental stem cells can be seeded in culture vessels at a density
that allows cell growth. For example, the cells may be seeded at low
density (e.g., about 1,000 to about 5,000 cells/cm2) to high density
(e.g., about 50,000 or more cells/cm2). In a preferred embodiment,
the cells are cultured at about 0 to about 5 percent by volume CO2
in air. In some preferred embodiments, the cells are cultured at about 2
to about 25 percent O2 in air, preferably about 5 to about 20
percent O2 in air. The cells preferably are cultured at about
25° C. to about 40° C., preferably 37° C. The cells
are preferably cultured in an incubator. The culture medium can be static
or agitated, for example, using a bioreactor. Placental stem cells
preferably are grown under low oxidative stress (e.g., with addition of
glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, or
the like).

[0172]Once 70%-90% confluence is obtained, the cells may be passaged. For
example, the cells can be enzymatically treated, e.g., trypsinized, using
techniques well-known in the art, to separate them from the tissue
culture surface. After removing the cells by pipetting and counting the
cells, about 20,000-100,000 stem cells, preferably about 50,000 stem
cells, are passaged to a new culture container containing fresh culture
medium. Typically, the new medium is the same type of medium from which
the stem cells were removed. The invention encompasses populations of
placental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.

[0173]5.3.3 Placental Stem Cell Populations

[0174]The invention provides populations of placental stem cells.
Placental stem cell population can be isolated directly from one or more
placentas; that is, the placental stem cell population can be a
population of placental cells comprising placental stem cells obtained
from, or contained within, perfusate, or obtained from, or contained
within, disrupted placental tissue, e.g., placental tissue digestate
(that is, the collection of cells obtained by enzymatic digestion of a
placenta or part thereof). Isolated placental stem cells of the invention
can also be cultured and expanded to produce placental stem cell
populations. Populations of placental cells comprising placental stem
cells can also be cultured and expanded to produce placental stem cell
populations.

[0176]The invention provides methods of producing isolated placental stem
cell population by, e.g., selecting placental stem cells, whether derived
from enzymatic digestion or perfusion, that express particular markers
and/or particular culture or morphological characteristics. In one
embodiment, for example, the invention provides a method of producing a
cell population comprising selecting placental cells that (a) adhere to a
substrate, and (b) express CD200 and HLA-G; and isolating said cells from
other cells to form a cell population. In another embodiment, the
invention provides a method of producing a cell population comprising
identifying placental cells that express CD200 and HLA-G, and isolating
said cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises selecting
placental cells that (a) adhere to a substrate, and (b) express CD73,
CD105, and CD200; and isolating said cells from other cells to form a
cell population. In another embodiment, the invention provides a method
of producing a cell population comprising identifying placental cells
that express CD73, CD105, and CD200, and isolating said cells from other
cells to form a cell population. In another embodiment, the method of
producing a cell population comprises selecting placental cells that (a)
adhere to a substrate and (b) express CD200 and OCT-4; and isolating said
cells from other cells to form a cell population. In another embodiment,
the invention provides a method of producing a cell population comprising
identifying placental cells that express CD200 and OCT-4, and isolating
said cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises selecting
placental cells that (a) adhere to a substrate, (b) express CD73 and
CD105, and (c) facilitate the formation of one or more embryoid-like
bodies in a population of placental cells comprising said stem cell when
said population is cultured under conditions that allow for the formation
of an embryoid-like body; and isolating said cells from other cells to
form a cell population. In another embodiment, the invention provides a
method of producing a cell population comprising identifying placental
cells that express CD73 and CD105, and facilitate the formation of one or
more embryoid-like bodies in a population of placental cells comprising
said stem cell when said population is cultured under conditions that
allow for the formation of an embryoid-like body, and isolating said
cells from other cells to form a cell population. In another embodiment,
the method of producing a cell population comprises selecting placental
cells that (a) adhere to a substrate, and (b) express CD73, CD105 and
HLA-G; and isolating said cells from other cells to form a cell
population. In another embodiment, the invention provides a method of
producing a cell population comprising identifying placental cells that
express CD73, CD105 and HLA-G, and isolating said cells from other cells
to form a cell population. In another embodiment, the method of producing
a cell population comprises selecting placental cells that (a) adhere to
a substrate, (b) express OCT-4, and (c) facilitate the formation of one
or more embryoid-like bodies in a population of placental cells
comprising said stem cell when said population is cultured under
conditions that allow for the formation of an embryoid-like body; and
isolating said cells from other cells to form a cell population. In
another embodiment, the invention provides a method of producing a cell
population comprising identifying placental cells that express OCT-4, and
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells comprising said stem cell when said
population is cultured under conditions that allow for the formation of
an embryoid-like body, and isolating said cells from other cells to form
a cell population.

[0177]Such cell populations can be used to treat any of the diseases or
conditions listed hereinbelow. Such cell populations can also be used to
assess populations of placental stem cells, e.g., as part of a quality
control method.

[0178]In any of the above embodiments, the method can additionally
comprise selecting placental cells that express ABC-p (a
placenta-specific ABC transporter protein; see, e.g., Allikmets et al.,
Cancer Res. 58(23):5337-9 (1998)). The method can also comprise selecting
cells exhibiting at least one characteristic specific to, e.g., a
mesenchymal stem cell, for example, expression of CD29, expression of
CD44, expression of CD90, or expression of a combination of the
foregoing.

[0179]In the above embodiments, the substrate can be any surface on which
culture and/or selection of cells, e.g., placental stem cells, can be
accomplished. Typically, the substrate is plastic, e.g., tissue culture
dish or multiwell plate plastic. Tissue culture plastic can be coated
with a biomolecule, e.g., laminin or fibronectin.

[0180]Cells, e.g., placental stem cells, can be selected for a placental
stem cell population by any means known in the art of cell selection. For
example, cells can be selected using an antibody or antibodies to one or
more cell surface markers, for example, in flow cytometry or FACS.
Selection can be accomplished using antibodies in conjunction with
magnetic beads. Antibodies that are specific for certain stem
cell-related markers are known in the art. For example, antibodies to
OCT-4 (Abcam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abcam), CD73 (BD
Biosciences Pharmingen, San Diego, Calif.), CD105 (Abcam; BioDesign
International, Saco, Me.), etc. Antibodies to other markers are also
available commercially, e.g., CD34, CD38 and CD45 are available from,
e.g., StemCell Technologies or BioDesign International.

[0181]The isolated placental stem cell population can comprise placental
cells that are not stem cells, or cells that are not placental cells.

[0183]In one, an isolated population of placental stem cells is combined
with a plurality of hematopoietic stem cells. Such hematopoietic stem
cells can be, for example, contained within unprocessed placental,
umbilical cord blood or peripheral blood; in total nucleated cells from
placental blood, umbilical cord blood or peripheral blood; in an isolated
population of CD34- cells from placental blood, umbilical cord blood
or peripheral blood; in unprocessed bone marrow; in total nucleated cells
from bone marrow; in an isolated population of CD34+ cells from bone
marrow, or the like.

5.4 Production of a Placental Stem Cell Bank

[0184]Stem cells from postpartum placentas can be cultured in a number of
different ways to produce a set of lots, e.g., a set of
individually-administrable doses, of placental stem cells. Such lots can,
for example, be obtained from stem cells from placental perfusate or from
enzyme-digested placental tissue. Sets of lots of placental stem cells,
obtained from a plurality of placentas, can be arranged in a bank of
placental stem cells for, e.g., long-term storage. Generally, adherent
stem cells are obtained from an initial culture of placental material to
form a seed culture, which is expanded under controlled conditions to
form populations of cells from approximately equivalent numbers of
doublings. Lots are preferably derived from the tissue of a single
placenta, but can be derived from the tissue of a plurality of placentas.

[0185]In one embodiment, stem cell lots are obtained as follows. Placental
tissue is first disrupted, e.g., by mincing, digested with a suitable
enzyme, e.g., collagenase (see Section 5.2.3, above). The placental
tissue preferably comprises, e.g., the entire amnion, entire chorion, or
both, from a single placenta, but can comprise only a part of either the
amnion or chorion. The digested tissue is cultured, e.g., for about 1-3
weeks, preferably about 2 weeks. After removal of non-adherent cells,
high-density colonies that form are collected, e.g., by trypsinization.
These cells are collected and resuspended in a convenient volume of
culture medium, and defined as Passage 0 cells.

[0186]Passage 0 cells are then used to seed expansion cultures. Expansion
cultures can be any arrangement of separate cell culture apparatuses,
e.g., a Cell Factory by NUNC®. Cells in the Passage 0 culture can be
subdivided to any degree so as to seed expansion cultures with, e.g.,
1×103, 2×103, 3×103, 4×103,
5×103, 6×103, 7×103, 8×103,
9×103, 1×104, 1×104, 2×104,
3×104, 4×104, 5×104, 6×104,
7×104, 8×104, 9×104, or
10×104 stem cells. Preferably, from about 2×104 to
about 3×104 Passage 0 cells are used to seed each expansion
culture. The number of expansion cultures can depend upon the number of
Passage 0 cells, and may be greater or fewer in number depending upon the
particular placenta(s) from which the stem cells are obtained.

[0187]Expansion cultures are grown until the density of cells in culture
reaches a certain value, e.g., about 1×105 cells/cm2.
Cells can either be collected and cryopreserved at this point, or
passaged into new expansion cultures as described above. Cells can be
passaged, e.g., 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 times prior to use. A record of the cumulative number of
population doublings is preferably maintained during expansion
culture(s). The cells from a Passage 0 culture can be expanded for 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38 or 40 doublings, or up to 60 doublings. Preferably, however, the
number of population doublings, prior to dividing the population of cells
into individual doses, is between about 15 and about 30, preferably about
20 doublings. The cells can be culture continuously throughout the
expansion process, or can be frozen at one or more points during
expansion.

[0188]Cells to be used for individual doses can be frozen, e.g.,
cryopreserved for later use. Individual doses can comprise, e.g., about 1
million to about 100 million cells per ml, and can comprise between about
106 and about 109 cells in total.

[0189]In a specific embodiment, of the method, Passage 0 cells are
cultured for a first number of doublings, e.g., approximately 4
doublings, then frozen in a first cell bank. Cells from the first cell
bank are frozen and used to seed a second cell bank, the cells of which
are expanded for a second number of doublings, e.g., about another eight
doublings. Cells at this stage are collected and frozen and used to seed
new expansion cultures that are allowed to proceed for a third number of
doublings, e.g., about eight additional doublings, bringing the
cumulative number of cell doublings to about 20. Cells at the
intermediate points in passaging can be frozen in units of about 100,000
to about 10 million cells per ml, preferably about 1 million cells per ml
for use in subsequent expansion culture. Cells at about 20 doublings can
be frozen in individual doses of between about 1 million to about 100
million cells per ml for administration or use in making a stem
cell-containing composition.

[0190]In one embodiment, therefore, the invention provides a method of
making a placental stem cell bank, comprising: expanding primary culture
placental stem cells from a human post-partum placenta for a first
plurality of population doublings; cryopreserving said placental stem
cells to form a Master Cell Bank; expanding a plurality of placental stem
cells from the Master Cell Bank for a second plurality of population
doublings; cryopreserving said placental stem cells to form a Working
Cell Bank; expanding a plurality of placental stem cells from the Working
Cell Bank for a third plurality of population doublings; and
cryopreserving said placental stem cells in individual doses, wherein
said individual doses collectively compose a placental stem cell bank. In
a specific embodiment, the total number of population doublings is about
20. In another specific embodiment, said first plurality of population
doublings is about four population doublings; said second plurality of
population doublings is about eight population doublings; and said third
plurality of population doublings is about eight population doublings. In
another specific embodiment, said primary culture placental stem cells
comprise placental stem cells from placental perfusate. In another
specific embodiment, said primary culture placental stem cells comprise
placental stem cells from digested placental tissue. In another specific
embodiment, said primary culture placental stem cells comprise placental
stem cells from placental perfusate and from digested placental tissue.
In another specific embodiment, all of said placental stem cells in said
placental stem cell primary culture are from the same placenta. In
another specific embodiment, the method further comprises the step of
selecting CD200+ or HLA-G+ placental stem cells from said
plurality of said placental stem cells from said Working Cell Bank to
form individual doses. In another specific embodiment, said individual
doses comprise from about 104 to about 105 placental stem
cells. In another specific embodiment, said individual doses comprise
from about 105 to about 106 placental stem cells. In another
specific embodiment, said individual doses comprise from about 106
to about 107 placental stem cells. In another specific embodiment,
said individual doses comprise from about 107 to about 108
placental stem cells.

[0191]In a preferred embodiment, the donor from which the placenta is
obtained (e.g., the mother) is tested for at least one pathogen. If the
mother tests positive for a tested pathogen, the entire lot from the
placenta is discarded. Such testing can be performed at any time during
production of placental stem cell lots, including before or after
establishment of Passage 0 cells, or during expansion culture. Pathogens
for which the presence is tested can include, without limitation,
hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human
immunodeficiency virus (types I and II), cytomegalovirus, herpesvirus,
and the like.

5.5 Differentiation of Placental Stem Cells

[0192]5.5.1 Induction Of Differentiation into Neuronal or Neurogenic Cells

[0193]Neuronal differentiation of placental stem cells can be
accomplished, for example, by placing placental stem cells in cell
culture conditions that induce differentiation into neurons. In an
example method, a neurogenic medium comprises DMEM/20% FBS and 1 mM
beta-mercaptoethanol; such medium can be replaced after culture for about
24 hours with medium consisting of DMEM and 1-10 mM betamercaptoethanol.
In another embodiment, the cells are contacted with DMEM/2% DMSO/200
μM butylated hydroxyanisole. In a specific embodiment, the
differentiation medium comprises serum-free DMEMIF-12, butylated
hydroxyanisole, potassium chloride, insulin, forskolin, valproic acid,
and hydrocortisone. In another embodiment, neuronal differentiation is
accomplished by plating placental stem cells on laminin-coated plates in
Neurobasal-A medium (Invitrogen, Carlsbad Calif.) containing B27
supplement and L-glutamine, optionally supplemented with bFGF and/or EGF.
Placental stem cells can also be induced to neural differentiation by
co-culture with neural cells, or culture in neuron-conditioned medium.

[0194]Neuronal differentiation can be assessed, e.g., by detection of
neuron-like morphology (e.g., bipolar cells comprising extended
processes) detection of the expression of e.g., nerve growth factor
receptor and neurofilament heavy chain genes by RT-PCR; or detection of
electrical activity, e.g., by patch-clamp. A placental stem cell is
considered to have differentiated into a neuronal cell when the cell
displays one or more of these characteristics.

[0195]5.5.2 Induction of Differentiation into Adipogenic Cells

[0196]Adipogenic differentiation of placental stem cells can be
accomplished, for example, by placing placental stem cells in cell
culture conditions that induce differentiation into adipocytes. A
preferred adipogenic medium comprises MSCGM (Cambrex) or DMEM
supplemented with 15% cord blood serum. In one embodiment, placental stem
cells are fed Adipogenesis Induction Medium (Cambrex) and cultured for 3
days (at 37° C., 5% CO2), followed by 1-3 days of culture in
Adipogenesis Maintenance Medium (Cambrex). After 3 complete cycles of
induction/maintenance, the cells are cultured for an additional 7 days in
adipogenesis maintenance medium, replacing the medium every 2-3 days.

[0198]A hallmark of adipogenesis is the development of multiple
intracytoplasmic lipid vesicles that can be easily observed using the
lipophilic stain oil red O. Expression of lipase and/or fatty acid
binding protein genes is confirmed by RT/PCR in placental stem cells that
have begun to differentiate into adipocytes. A placental stem cell is
considered to have differentiated into an adipocytic cell when the cell
displays one or more of these characteristics.

[0199]5.5.3 Induction of Differentiation into Chondrocytic Cells

[0200]Chondrogenic differentiation of placental stem cells can be
accomplished, for example, by placing placental stem cells in cell
culture conditions that induce differentiation into chondrocytes. A
preferred chondrocytic medium comprises MSCGM (Cambrex) or DMEM
supplemented with 15% cord blood serum. In one embodiment, placental stem
cells are aliquoted into a sterile polypropylene tube, centrifuged (e.g.,
at 150×g for 5 minutes), and washed twice in Incomplete
Chondrogenesis Medium (Cambrex). The cells are resuspended in Complete
Chondrogenesis Medium (Cambrex) containing 0.01 μg/ml TGF-beta-3 at a
concentration of about 1-20×105 cells/ml. In other
embodiments, placental stem cells are contacted with exogenous growth
factors, e.g., GDF-5 or transforming growth factor beta3 (TGF-beta3),
with or without ascorbate. Chondrogenic medium can be supplemented with
amino acids including proline and glutamine, sodium pyruvate,
dexamethasone, ascorbic acid, and insulin/transferrin/selenium.
Chondrogenic medium can be supplemented with sodium hydroxide and/or
collagen. The placental stem cells may be cultured at high or low
density. Cells are preferably cultured in the absence of serum.

[0201]Chondrogenesis can be assessed by e.g., observation of production of
esoinophilic ground substance, safranin-O staining for glycosaminoglycan
expression; hematoxylin/eosin staining, assessing cell morphology, and/or
RT/PCR confirmation of collagen 2 and collagen 9 gene expression.
Chondrogenesis can also be observed by growing the stem cells in a
pellet, formed, e.g., by gently centrifuging stem cells in suspension
(e.g., at about 800 g for about 5 minutes). After about 1-28 days, the
pellet of stem cells begins to form a tough matrix and demonstrates a
structural integrity not found in non-induced, or non-chondrogenic, cell
lines, pellets of which tend to fall apart when challenged.
Chondrogenesis can also be demonstrated, e.g., in such cell pellets, by
staining with a stain that stains collage, e.g., Sirius Red, and/or a
stain that stains glycosaminoglycans (GAGs), such as, e.g., Alcian Blue.
A placental stem cell is considered to have differentiated into a
chondrocytic cell when the cell displays one or more of these
characteristics.

[0204]Differentiation can be assayed using a calcium-specific stain, e.g.,
von Kossa staining, and RT/PCR detection of, e.g., alkaline phosphatase,
osteocalcin, bone sialoprotein and/or osteopontin gene expression. A
placental stem cell is considered to have differentiated into an
osteocytic cell when the cell displays one or more of these
characteristics.

[0205]5.5.5 Induction of Differentiation into Pancreatic Cells

[0206]Differentiation of placental stem cells into insulin-producing
pancreatic cells can be accomplished, for example, by placing placental
stem cells in cell culture conditions that induce differentiation into
pancreatic cells.

[0208]Differentiation can be confirmed by assaying for, e.g., insulin
protein production, or insulin gene expression by RT/PCR. A placental
stem cell is considered to have differentiated into a pancreatic cell
when the cell displays one or more of these characteristics.

[0211]Differentiation can be confirmed by demonstration of cardiac actin
gene expression, e.g., by RT/PCR, or by visible beating of the cell. A
placental stem cell is considered to have differentiated into a cardiac
cell when the cell displays one or more of these characteristics.

5.6 Preservation of Placental Stem Cells

[0212]Placental stem cells can be preserved, that is, placed under
conditions that allow for long-term storage, or conditions that inhibit
cell death by, e.g., apoptosis or necrosis.

[0213]Placental stem cells can be preserved using, e.g., a composition
comprising an apoptosis inhibitor, necrosis inhibitor and/or an
oxygen-carrying perfluorocarbon, as described in related U.S. Provisional
Application No. 60/754,969, entitled "Improved Medium for Collecting
Placental Stem Cells and Preserving Organs," filed on Dec. 25, 2005. In
one embodiment, the invention provides a method of preserving a
population of stem cells comprising contacting said population of stem
cells with a stem cell collection composition comprising an inhibitor of
apoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitor
of apoptosis is present in an amount and for a time sufficient to reduce
or prevent apoptosis in the population of stem cells, as compared to a
population of stem cells not contacted with the inhibitor of apoptosis.
In a specific embodiment, said inhibitor of apoptosis is a caspase
inhibitor. In another specific embodiment, said inhibitor of apoptosis is
a JNK inhibitor. In a more specific embodiment, said JNK inhibitor does
not modulate differentiation or proliferation of said stem cells. In
another embodiment, said stem cell collection composition comprises said
inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in
separate phases. In another embodiment, said stem cell collection
composition comprises said inhibitor of apoptosis and said
oxygen-carrying perfluorocarbon in an emulsion. In another embodiment,
the stem cell collection composition additionally comprises an
emulsifier, e.g., lecithin. In another embodiment, said apoptosis
inhibitor and said perfluorocarbon are between about 0° C. and
about 25° C. at the time of contacting the stem cells. In another
more specific embodiment, said apoptosis inhibitor and said
perfluorocarbon are between about 2° C. and 10° C., or
between about 2° C. and about 5° C., at the time of
contacting the stem cells. In another more specific embodiment, said
contacting is performed during transport of said population of stem
cells. In another more specific embodiment, said contacting is performed
during freezing and thawing of said population of stem cells.

[0214]In another embodiment, the invention provides a method of preserving
a population of placental stem cells comprising contacting said
population of stem cells with an inhibitor of apoptosis and an
organ-preserving compound, wherein said inhibitor of apoptosis is present
in an amount and for a time sufficient to reduce or prevent apoptosis in
the population of stem cells, as compared to a population of stem cells
not contacted with the inhibitor of apoptosis. In a specific embodiment,
the organ-preserving compound is UW solution (described in U.S. Pat. No.
4,798,824; also known as ViaSpan; see also Southard et al.,
Transplantation 49(2):251-257 (1990)) or a solution described in Stern et
al., U.S. Pat. No. 5,552,267. In another embodiment, said
organ-preserving compound is hydroxyethyl starch, lactobionic acid,
raffinose, or a combination thereof. In another embodiment, the stem cell
collection composition additionally comprises an oxygen-carrying
perfluorocarbon, either in two phases or as an emulsion.

[0215]In another embodiment of the method, placental stem cells are
contacted with a stem cell collection composition comprising an apoptosis
inhibitor and oxygen-carrying perfluorocarbon, organ-preserving compound,
or combination thereof, during perfusion. In another embodiment, said
stem cells are contacted during a process of tissue disruption, e.g.,
enzymatic digestion. In another embodiment, placental stem cells are
contacted with said stem cell collection compound after collection by
perfusion, or after collection by tissue disruption, e.g., enzymatic
digestion.

[0216]Typically, during placental cell collection, enrichment and
isolation, it is preferable to minimize or eliminate cell stress due to
hypoxia and mechanical stress. In another embodiment of the method,
therefore, a stem cell, or population of stem cells, is exposed to a
hypoxic condition during collection, enrichment or isolation for less
than six hours during said preservation, wherein a hypoxic condition is a
concentration of oxygen that is less than normal blood oxygen
concentration. In a more specific embodiment, said population of stem
cells is exposed to said hypoxic condition for less than two hours during
said preservation. In another more specific embodiment, said population
of stem cells is exposed to said hypoxic condition for less than one
hour, or less than thirty minutes, or is not exposed to a hypoxic
condition, during collection, enrichment or isolation. In another
specific embodiment, said population of stem cells is not exposed to
shear stress during collection, enrichment or isolation.

[0217]The placental stem cells of the invention can be cryopreserved,
e.g., in cryopreservation medium in small containers, e.g., ampoules.
Suitable cryopreservation medium includes, but is not limited to, culture
medium including, e.g., growth medium, or cell freezing medium, for
example commercially available cell freezing medium, e.g., C2695, C2639
or C6039 (Sigma). Cryopreservation medium preferably comprises DMSO
(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).
Cryopreservation medium may comprise additional agents, for example,
methylcellulose and/or glycerol. Placental stem cells are preferably
cooled at about 1 ° C/min during cryopreservation. A preferred
cryopreservation temperature is about -80° C. to about
-180° C., preferably about -125° C. to about -140°
C. Cryopreserved cells can be transferred to liquid nitrogen prior to
thawing for use. In some embodiments, for example, once the ampoules have
reached about -90° C., they are transferred to a liquid nitrogen
storage area. Cryopreservation can also be done using a controlled-rate
freezer. Cryopreserved cells preferably are thawed at a temperature of
about 25° C. to about 40° C., preferably to a temperature
of about 37° C.

5.7 Uses of Placental Stem Cells

[0218]5.7.1 Placental Stem Cell Populations

[0219]Placental stem cell populations can be used to treat any disease,
disorder or condition that is amenable to treatment by administration of
a population of stem cells. As used herein, "treat" encompasses the cure
of, remediation of, improvement of, lessening of the severity of, or
reduction in the time course of, a disease, disorder or condition, or any
parameter or symptom thereof.

[0224]In other embodiments, isolated populations of placental stem cells
may be used in autologous or heterologous tissue regeneration or
replacement therapies or protocols, including, but not limited to
treatment of corneal epithelial defects, treatment of osteogenesis
imperfecta, cartilage repair, facial dermabrasion, mucosal membranes,
tympanic membranes, intestinal linings, neurological structures (e.g.,
retina, auditory neurons in basilar membrane, olfactory neurons in
olfactory epithelium), burn and wound repair for traumatic injuries of
the skin, or for reconstruction of other damaged or diseased organs or
tissues.

[0225]In a preferred embodiment, an isolated population of placental stem
cells is used in hematopoietic reconstitution in an individual that has
suffered a partial or total loss of hematopoietic stem cells, e.g.,
individuals exposed to lethal or sub-lethal doses of radiation (whether
industrial, medical or military); individuals that have undergone
myeloablation as part of, e.g., cancer therapy, and the like, in the
treatment of, e.g., a hematologic malignancy. Placental stem cells can be
used in hematopoietic reconstitution in individuals having anemia (e.g.,
aplastic anemia, sickle cell anemia, etc.). Preferably, the placental
stem cells are administered to such individuals with a population of
hematopoietic stem cells. Isolated populations of placental-derived stem
cells can be used in place of, or to supplement, bone marrow or
populations of stem cells derived from bone marrow. Typically,
approximately 1×108 to 2×108 bone marrow
mononuclear cells per kilogram of patient weight are infused for
engraftment in a bone marrow transplantation (i.e., about 70 ml of marrow
for a 70 kg donor). To obtain 70 ml requires an intensive donation and
significant loss of donor blood in the donation process. An isolated
population of placental stem cells for hematopoietic reconstitution can
comprise, in various embodiments, about, at least, or no more than
1×105, 5×105, 1×106, 5×106,
1×107, 5×107, 1×108, 1×109,
1×1010, 5×1010, 1×1011 or more placental
stem cells.

[0226]In one embodiment, therefore, placental stem cells can be used to
treat patients having a blood cancer, such as a lymphoma, leukemia (such
as chronic or acute myelogenous leukemia, acute lymphocytic leukemia,
Hodgkin's disease, etc.), myelodysplasia, myelodysplastic syndrome, and
the like. In another embodiment, the disease, disorder or condition is
chronic granulomatous disease.

[0227]Because hematopoietic reconstitution can be used in the treatment of
anemias, the present invention further encompasses the treatment of an
individual with a stem cell combination of the invention, wherein the
individual has an anemia or disorder of the blood hemoglobin. The anemia
or disorder may be natural (e.g., caused by genetics or disease), or may
be artificially-induced (e.g., by accidental or deliberate poisoning,
chemotherapy, and the like). In another embodiment, the disease or
disorder is a marrow failure syndrome (e.g., aplastic anemia, Kostmann
syndrome, Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, and
the like), a bone marrow disorder or a hematopoietic disease or disorder.

[0228]Placental stem cells can also be used to treat severe combined
immunodeficiency disease, including, but not limited to, combined
immunodeficiency disease (e.g., Wiskott-Aldrich syndrome, severe DiGeorge
syndrome, and the like).

[0229]The placental stem cells of the invention, alone or in combination
with other stem cell or progenitor cell populations, can be used in the
manufacture of a tissue or organ in vivo. The methods of the invention
encompass using cells obtained from the placenta, e.g., stem cells or
progenitor cells, to seed a matrix and to be cultured under the
appropriate conditions to allow the cells to differentiate and populate
the matrix. The tissues and organs obtained by the methods of the
invention can be used for a variety of purposes, including research and
therapeutic purposes.

[0230]In a preferred embodiment of the invention, placental stem cells and
placental stem cell populations may be used for autologous and allogenic
transplants, including matched and mismatched HLA type hematopoietic
transplants. In one embodiment of the use of placental stem cells as
allogenic hematopoietic transplants, the host is treated to reduce
immunological rejection of the donor cells, or to create immunotolerance
(see, e.g., U.S. Pat. Nos. 5,800,539 and 5,806,529). In another
embodiment, the host is not treated to reduce immunological rejection or
to create immunotolerance.

[0231]Placental stem cells, either alone or in combination with one or
more other stem cell populations, can be used in therapeutic
transplantation protocols, e.g., to augment or replace stem or progenitor
cells of the liver, pancreas, kidney, lung, nervous system, muscular
system, bone, bone marrow, thymus, spleen, mucosal tissue, gonads, or
hair. Additionally, placental stem cells may be used instead of specific
classes of progenitor cells (e.g., chondrocytes, hepatocytes,
hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscle
progenitor cells, etc.) in therapeutic or research protocols in which
progenitor cells would typically be used.

[0232]In one embodiment, the invention provides for the use of placental
stem cells, particularly CD200- placental stem cells, as an adjunct
to hair replacement therapy. For example, in one embodiment, placental
stem cells, e.g., CD200- placental stem cells, are injected
subcutaneously or intradermally at a site in which hair growth or
regrowth is desired. The number of stem cells injected can be, e.g.,
between about 100 and about 10,000 per injection, in a volume of about
0.1 to about 1.0 μL, though more ore fewer cells in a greater or
lesser volume can also be used. Administration of placental stem cells to
facilitate hair regrowth can comprise a single injection or multiple
injections in, e.g., a regular or a random pattern in an area in which
hair regrowth is desired. Known hair regrowth therapies can be used in
conjunction with the placental stem cells, e.g., topical minoxidil. Hair
loss that can be treated using placental stem cells can be
naturally-occurring (e.g., male pattern baldness) or induced (e.g.,
resulting from toxic chemical exposure).

[0233]Placental stem cells and placental stem cell populations of the
invention can be used for augmentation, repair or replacement of
cartilage, tendon, or ligaments. For example, in certain embodiments,
prostheses (e.g., hip prostheses) can be coated with replacement
cartilage tissue constructs grown from placental stem cells of the
invention. In other embodiments, joints (e.g., knee) can be reconstructed
with cartilage tissue constructs grown from placental stem cells.
Cartilage tissue constructs can also be employed in major reconstructive
surgery for different types of joints (see, e.g., Resnick & Niwayama,
eds., 1988, Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders
Co.).

[0234]The placental stem cells of the invention can be used to repair
damage to tissues and organs resulting from, e.g., trauma, metabolic
disorders, or disease. The trauma can be, e.g., trauma from surgery,
e.g., cosmetic surgery. In such an embodiment, a patient can be
administered placental stem cells, alone or combined with other stem or
progenitor cell populations, to regenerate or restore tissues or organs
which have been damaged as a consequence of disease.

[0235]5.7.2 Compositions Comprising Placental Stem Cells

[0236]The present invention provides compositions comprising placental
stem cells, or biomolecules therefrom. The placental stem cells of the
present invention can be combined with any physiologically-acceptable or
medically-acceptable compound, composition or device for use in, e.g.,
research or therapeutics.

[0237]5.7.2.1 Cryopreserved Placental Stem Cells

[0238]The placental stem cell populations of the invention can be
preserved, for example, cryopreserved for later use. Methods for
cryopreservation of cells, such as stem cells, are well known in the art.
Placental stem cell populations can be prepared in a form that is easily
administrable to an individual. For example, the invention provides a
placental stem cell population that is contained within a container that
is suitable for medical use. Such a container can be, for example, a
sterile plastic bag, flask, jar, or other container from which the
placental stem cell population can be easily dispensed. For example, the
container can be a blood bag or other plastic, medically-acceptable bag
suitable for the intravenous administration of a liquid to a recipient.
The container is preferably one that allows for cryopreservation of the
combined stem cell population.

[0239]The cryopreserved placental stem cell population can comprise
placental stem cells derived from a single donor, or from multiple
donors. The placental stem cell population can be completely HLA-matched
to an intended recipient, or partially or completely HLA-mismatched.

[0240]Thus, in one embodiment, the invention provides a composition
comprising a placental stem cell population in a container. In a specific
embodiment, the stem cell population is cryopreserved. In another
specific embodiment, the container is a bag, flask, or jar. In more
specific embodiment, said bag is a sterile plastic bag. In a more
specific embodiment, said bag is suitable for, allows or facilitates
intravenous administration of said placental stem cell population. The
bag can comprise multiple lumens or compartments that are interconnected
to allow mixing of the placental stem cells and one or more other
solutions, e.g., a drug, prior to, or during, administration. In another
specific embodiment, the composition comprises one or more compounds that
facilitate cryopreservation of the combined stem cell population. In
another specific embodiment, said placental stem cell population is
contained within a physiologically-acceptable aqueous solution. In a more
specific embodiment, said physiologically-acceptable aqueous solution is
a 0.9% NaCl solution. In another specific embodiment, said placental stem
cell population comprises placental cells that are HLA-matched to a
recipient of said stem cell population. In another specific embodiment,
said combined stem cell population comprises placental cells that are at
least partially HLA-mismatched to a recipient of said stem cell
population. In another specific embodiment, said placental stem cells are
derived from a plurality of donors.

[0241]5.7.2.2 Pharmaceutical Compositions

[0242]Populations of placental stem cells, or populations of cells
comprising placental stem cells, can be formulated into pharmaceutical
compositions for use in vivo. Such pharmaceutical compositions comprise a
population of placental stem cells, or a population of cells comprising
placental stem cells, in a pharmaceutically-acceptable carrier, e.g., a
saline solution or other accepted physiologically-acceptable solution for
in vivo administration. Pharmaceutical compositions of the invention can
comprise any of the placental stem cell populations, or placental stem
cell types, described elsewhere herein. The pharmaceutical compositions
can comprise fetal, maternal, or both fetal and maternal placental stem
cells. The pharmaceutical compositions of the invention can further
comprise placental stem cells obtained from a single individual or
placenta, or from a plurality of individuals or placentae.

[0243]The pharmaceutical compositions of the invention can comprise any
number of placental stem cells. For example, a single unit dose of
placental stem cells can comprise, in various embodiments, about, at
least, or no more than 1×105, 5×105,
1×106, 5×106, 1×107, 5×107,
1×108, 5×108, 1×109, 5×109,
1×1010, 5×1010, 1×1011 or more placental
stem cells.

[0244]The pharmaceutical compositions of the invention comprise
populations of cells that comprise 50% viable cells or more (that is, at
least 50% of the cells in the population are functional or living).
Preferably, at least 60% of the cells in the population are viable. More
preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the
population in the pharmaceutical composition are viable.

[0245]The pharmaceutical compositions of the invention can comprise one or
more compounds that, e.g., facilitate engraftment (e.g., anti-T-cell
receptor antibodies, an immunosuppressant, or the like); stabilizers such
as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

[0246]When formulated as an injectable solution, in one embodiment, the
pharmaceutical composition of the invention comprises about 1.25% HSA and
about 2.5% dextran. Other injectable formulations, suitable for the
administration of cellular products, may be used.

[0247]In one embodiment, the composition of the invention comprises
placental stem cells that are substantially, or completely, non-maternal
in origin. For example, the invention provides in one embodiment a
composition comprising a population of placental stem cells that are
CD200+ and HLA-G+; CD73+, CD105+, and CD200+;
CD200+ and OCT-4+; CD73+; CD105+ and HLA-G+;
CD73+ and CD105+ and facilitate the formation of one or more
embryoid-like bodies in a population of placental cells comprising said
population of placental stem cell when said population of placental cells
is cultured under conditions that allow the formation of an embryoid-like
body; or OCT-4+ and facilitate the formation of one or more
embryoid-like bodies in a population of placental cells comprising said
population of placental stem cell when said population of placental cells
is cultured under conditions that allow the formation of an embryoid-like
body; or a combination of the foregoing, wherein at least 70%, 80%, 90%,
95% or 99% of said placental stem cells are non-maternal in origin. In a
specific embodiment, the composition additionally comprises a stem cell
that is not obtained from a placenta.

[0248]5.7.2.3 Placental Stem Cell Conditioned Media

[0249]The placental stem cells of the invention can be used to produce
conditioned medium, that is, medium comprising one or more biomolecules
secreted or excreted by the stem cells. In various embodiments, the
conditioned medium comprises medium in which placental stem cells have
grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more
days. In other embodiments, the conditioned medium comprises medium in
which placental stem cells have grown to at least 30%, 40%, 50%, 60%,
70%, 80%, 90% confluence, or up to 100% confluence. Such conditioned
medium can be used to support the culture of a separate population of
placental stem cells, or stem cells of another kind In another
embodiment, the conditioned medium comprises medium in which placental
stem cells have been differentiated into an adult cell type. In another
embodiment, the conditioned medium of the invention comprises medium in
which placental stem cells and non-placental stem cells have been
cultured.

[0250]5.7.2.4 Matrices Comprising Placental Stem Cells

[0251]The invention further comprises matrices, hydrogels, scaffolds, and
the like that comprise a placental stem cell, or a population of
placental stem cells.

[0252]Placental stem cells of the invention can be seeded onto a natural
matrix, e.g., a placental biomaterial such as an amniotic membrane
material. Such an amniotic membrane material can be, e.g., amniotic
membrane dissected directly from a mammalian placenta; fixed or
heat-treated amniotic membrane, substantially dry (i.e., <20%
H2O) amniotic membrane, chorionic membrane, substantially dry
chorionic membrane, substantially dry amniotic and chorionic membrane,
and the like. Preferred placental biomaterials on which placental stem
cells can be seeded are described in Hariri, U.S. Application Publication
No. 2004/0048796.

[0253]Placental stem cells of the invention can be suspended in a hydrogel
solution suitable for, e.g., injection. Suitable hydrogels for such
compositions include self-assembling peptides, such as RAD16. In one
embodiment, a hydrogel solution comprising the cells can be allowed to
harden, for instance in a mold, to form a matrix having cells dispersed
therein for implantation. Placental stem cells in such a matrix can also
be cultured so that the cells are mitotically expanded prior to
implantation. The hydrogel is, e.g., an organic polymer (natural or
synthetic) that is cross-linked via covalent, ionic, or hydrogen bonds to
create a three-dimensional open-lattice structure that entraps water
molecules to form a gel. Hydrogel-forming materials include
polysaccharides such as alginate and salts thereof, peptides,
polyphosphazines, and polyacrylates, which are crosslinked ionically, or
block polymers such as polyethylene oxide-polypropylene glycol block
copolymers which are crosslinked by temperature or pH, respectively. In
some embodiments, the hydrogel or matrix of the invention is
biodegradable.

[0255]In some embodiments, the polymers are at least partially soluble in
aqueous solutions, such as water, buffered salt solutions, or aqueous
alcohol solutions, that have charged side groups, or a monovalent ionic
salt thereof. Examples of polymers having acidic side groups that can be
reacted with cations are poly(phosphazenes), poly(acrylic acids),
poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid,
poly(vinyl acetate), and sulfonated polymers, such as sulfonated
polystyrene. Copolymers having acidic side groups formed by reaction of
acrylic or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups, sulfonic
acid groups, halogenated (preferably fluorinated) alcohol groups,
phenolic OH groups, and acidic OH groups.

[0256]The placental stem cells of the invention or co-cultures thereof can
be seeded onto a three-dimensional framework or scaffold and implanted in
vivo. Such a framework can be implanted in combination with any one or
more growth factors, cells, drugs or other components that stimulate
tissue formation or otherwise enhance or improve the practice of the
invention.

[0257]Examples of scaffolds that can be used in the present invention
include nonwoven mats, porous foams, or self assembling peptides.
Nonwoven mats can be formed using fibers comprised of a synthetic
absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA)
(VICRYL, Ethicon, Inc., Somerville, N.J.). Foams, composed of, e.g.,
poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,
formed by processes such as freeze-drying, or lyophilization (see, e.g.,
U.S. Pat. No. 6,355,699), can also be used as scaffolds.

[0259]In another embodiment, placental stem cells can be seeded onto, or
contacted with, a felt, which can be, e.g., composed of a multifilament
yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers
or blends, or hyaluronic acid.

[0260]The placental stem cells of the invention can, in another
embodiment, be seeded onto foam scaffolds that may be composite
structures. Such foam scaffolds can be molded into a useful shape, such
as that of a portion of a specific structure in the body to be repaired,
replaced or augmented. In some embodiments, the framework is treated,
e.g., with 0.1M acetic acid followed by incubation in polylysine, PBS,
and/or collagen, prior to inoculation of the cells of the invention in
order to enhance cell attachment. External surfaces of a matrix may be
modified to improve the attachment or growth of cells and differentiation
of tissue, such as by plasma-coating the matrix, or addition of one or
more proteins (e.g., collagens, elastic fibers, reticular fibers),
glycoproteins, glycosaminoglycans (e.g., heparin sulfate,
chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin
sulfate, etc.), a cellular matrix, and/or other materials such as, but
not limited to, gelatin, alginates, agar, agarose, and plant gums, and
the like.

[0261]In some embodiments, the scaffold comprises, or is treated with,
materials that render it non-thrombogenic. These treatments and materials
may also promote and sustain endothelial growth, migration, and
extracellular matrix deposition. Examples of these materials and
treatments include but are not limited to natural materials such as
basement membrane proteins such as laminin and Type IV collagen,
synthetic materials such as EPTFE, and segmented polyurethaneurea
silicones, such as PURSPAN® (The Polymer Technology Group, Inc.,
Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agents
such as heparin; the scaffolds can also be treated to alter the surface
charge (e.g., coating with plasma) prior to seeding with placental stem
cells.

[0262]5.7.3 Immortalized Placental Stem Cell Lines

[0263]Mammalian placental cells can be conditionally immortalized by
transfection with any suitable vector containing a growth-promoting gene,
that is, a gene encoding a protein that, under appropriate conditions,
promotes growth of the transfected cell, such that the production and/or
activity of the growth-promoting protein is regulatable by an external
factor. In a preferred embodiment the growth-promoting gene is an
oncogene such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40
large T antigen, polyoma large T antigen, Ela adenovirus or E7 protein of
human papillomavirus.

[0264]External regulation of the growth-promoting protein can be achieved
by placing the growth-promoting gene under the control of an
externally-regulatable promoter, e.g., a promoter the activity of which
can be controlled by, for example, modifying the temperature of the
transfected cells or the composition of the medium in contact with the
cells. in one embodiment, a tetracycline (tet)-controlled gene expression
system can be employed (see Gossen et al., Proc. Natl. Acad. Sci. USA
89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci. USA
93:1518-1523, 1996). In the absence of tet, a tet-controlled
transactivator (tTA) within this vector strongly activates transcription
from phCMV*-1, a minimal promoter from human cytomegalovirus fused
to tet operator sequences. tTA is a fusion protein of the repressor
(tetR) of the transposon-10-derived tet resistance operon of Escherichia
coli and the acidic domain of VP16 of herpes simplex virus. Low,
non-toxic concentrations of tet (e.g., 0.01-1.0 μg/mL) almost
completely abolish transactivation by tTA.

[0265]In one embodiment, the vector further contains a gene encoding a
selectable marker, e.g., a protein that confers drug resistance. The
bacterial neomycin resistance gene (neoR) is one such marker that
may be employed within the present invention. Cells carrying neoR
may be selected by means known to those of ordinary skill in the art,
such as the addition of, e.g., 100-200 μg/mL G418 to the growth
medium.

[0266]Transfection can be achieved by any of a variety of means known to
those of ordinary skill in the art including, but not limited to,
retroviral infection. In general, a cell culture may be transfected by
incubation with a mixture of conditioned medium collected from the
producer cell line for the vector and DMEM/F12 containing N2 supplements.
For example, a placental cell culture prepared as described above may be
infected after, e.g., five days in vitro by incubation for about 20 hours
in one volume of conditioned medium and two volumes of DMEM/F12
containing N2 supplements. Transfected cells carrying a selectable marker
may then be selected as described above.

[0267]Following transfection, cultures are passaged onto a surface that
permits proliferation, e.g., allows at least 30% of the cells to double
in a 24 hour period. Preferably, the substrate is a polyornithine/laminin
substrate, consisting of tissue culture plastic coated with polyornithine
(10 μg/mL) and/or laminin (10 μg/mL), a polylysine/laminin
substrate or a surface treated with fibronectin. Cultures are then fed
every 3-4 days with growth medium, which may or may not be supplemented
with one or more proliferation-enhancing factors. Proliferation-enhancing
factors may be added to the growth medium when cultures are less than 50%
confluent.

[0268]The conditionally-immortalized placental stem cell lines can be
passaged using standard techniques, such as by trypsinization, when
80-95% confluent. Up to approximately the twentieth passage, it is, in
some embodiments, beneficial to maintain selection (by, for example, the
addition of G418 for cells containing a neomycin resistance gene). Cells
may also be frozen in liquid nitrogen for long-term storage.

[0269]Clonal cell lines can be isolated from a conditionally-immortalized
human placental stem cell line prepared as described above. In general,
such clonal cell lines may be isolated using standard techniques, such as
by limit dilution or using cloning rings, and expanded. Clonal cell lines
may generally be fed and passaged as described above.

[0270]Conditionally-immortalized human placental stem cell lines, which
may, but need not, be clonal, may generally be induced to differentiate
by suppressing the production and/or activity of the growth-promoting
protein under culture conditions that facilitate differentiation. For
example, if the gene encoding the growth-promoting protein is under the
control of an externally-regulatable promoter, the conditions, e.g.,
temperature or composition of medium, may be modified to suppress
transcription of the growth-promoting gene. For the
tetracycline-controlled gene expression system discussed above,
differentiation can be achieved by the addition of tetracycline to
suppress transcription of the growth-promoting gene. In general, 1
μg/mL tetracycline for 4-5 days is sufficient to initiate
differentiation. To promote further differentiation, additional agents
may be included in the growth medium.

[0271]5.7.4 Assays

[0272]The placental stem cells for the present invention can be used in
assays to determine the influence of culture conditions, environmental
factors, molecules (e.g., biomolecules, small inorganic molecules. etc.)
and the like on stem cell proliferation, expansion, and/or
differentiation, compared to placental stem cells not exposed to such
conditions.

[0273]In a preferred embodiment, the placental stem cells of the present
invention are assayed for changes in proliferation, expansion or
differentiation upon contact with a molecule. In one embodiment, for
example, the invention provides a method of identifying a compound that
modulates the proliferation of a plurality of placental stem cells,
comprising contacting said plurality of stem cells with said compound
under conditions that allow proliferation, wherein if said compound
causes a detectable change in proliferation of said plurality of stem
cells compared to a plurality of stem cells not contacted with said
compound, said compound is identified as a compound that modulates
proliferation of placental stem cells. In a specific embodiment, said
compound is identified as an inhibitor of proliferation. In another
specific embodiment, said compound is identified as an enhancer of
proliferation.

[0274]In another embodiment, the invention provides a method of
identifying a compound that modulates the expansion of a plurality of
placental stem cells, comprising contacting said plurality of stem cells
with said compound under conditions that allow expansion, wherein if said
compound causes a detectable change in expansion of said plurality of
stem cells compared to a plurality of stem cells not contacted with said
compound, said compound is identified as a compound that modulates
expansion of placental stem cells. In a specific embodiment, said
compound is identified as an inhibitor of expansion. In another specific
embodiment, said compound is identified as an enhancer of expansion.

[0275]In another embodiment, the invention provides a method of
identifying a compound that modulates the differentiation of a placental
stem cell, comprising contacting said stem cells with said compound under
conditions that allow differentiation, wherein if said compound causes a
detectable change in differentiation of said stem cells compared to a
stem cell not contacted with said compound, said compound is identified
as a compound that modulates proliferation of placental stem cells. In a
specific embodiment, said compound is identified as an inhibitor of
differentiation. In another specific embodiment, said compound is
identified as an enhancer of differentiation.

[0277]The culture flask in which the cells are cultured is prepared as
follows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBS
containing 5 ng/ml human FN (Sigma F0895) to the flask. The flasks with
FN solution are left at 37° C. for 30 min. The FN solution is then
removed prior to cell culture. There is no need to dry the flasks
following treatment. Alternatively, the flasks are left in contact with
the FN solution at 4° C. overnight or longer; prior to culture,
the flasks are warmed and the FN solution is removed.

[0278]Placental Stem Cells Isolated by Perfusion

[0279]Cultures of placental stem cells from placental perfusate are
established as follows. Cells from a Ficoll gradient are seeded in
FN-coated T75 flasks, prepared as above, at 50-100×106
cells/flask in 15 ml culture medium. Typically, 5 to 10 flasks are
seeded. The flasks are incubated at 37° C. for 12-18 hrs to allow
the attachment of adherent cells. 10 ml of warm PBS is added to each
flask to remove cells in suspension, and mixed gently. 15 mL of the
medium is then removed and replaced with 15 ml fresh culture medium. All
medium is changed 3-4 days after the start of culture. Subsequent culture
medium changes are performed, during which 50% or 7.5 ml of the medium is
removed.

[0280]Starting at about day 12, the culture is checked under a microscope
to examine the growth of the adherent cell colonies. When cell cultures
become approximately 80% confluent, typically between day 13 to day 18
after the start of culture, adherent cells are harvested by trypsin
digestion. Cells harvested from these primary cultures are designated
passage 0 (zero).

[0282]Placental stem cell cultures are established from digested placental
tissue as follows. The perfused placenta is placed on a sterile paper
sheet with the maternal side up. Approximately 0.5 cm of the surface
layer on maternal side of placenta is scraped off with a blade, and the
blade is used to remove a placental tissue block measuring approximately
1×2×1 cm. This placenta tissue is then minced into
approximately 1 mm3 pieces. These pieces are collected into a 50 ml
Falcon tube and digested with collagenase IA (2 mg/ml, Sigma) for 30
minutes, followed by trypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at
37° C. in water bath. The resulting solution is centrifuged at 400
g for 10 minutes at room temperature, and the digestion solution is
removed. The pellet is resuspended to approximately 10 volumes with PBS
(for example, a 5 ml pellet is resuspended with 45 ml PBS), and the tubes
are centrifuged at 400 g for 10 minutes at room temperature. The
tissue/cell pellet is resuspended in 130 mL culture medium, and the cells
are seeded at 13 ml per fibronectin-coated T-75 flask. Cells are
incubated at 37° C. with a humidified atmosphere with 5% CO2.
Placental Stem Cells are optionally cryopreserved at this stage.

[0283]Subculturing and Expansion of Placental Stem Cells

[0284]Cryopreserved cells are quickly thawed in a 37° C. water
bath. Placental stem cells are immediately removed from the cryovial with
10 ml warm medium and transferred to a 15 ml sterile tube. The cells are
centrifuged at 400 g for 10 minutes at room temperature. The cells are
gently resuspended in 10 ml of warm culture medium by pipetting, and
viable cell counts are determined by Trypan blue exclusion. Cells are
then seeded at about 6000-7000 cells per cm2 onto FN-coated flasks,
prepared as above (approximately 5×105 cells per T-75 flask).
The cells are incubated at 37° C., 5% CO2 and 90% humidity.
When the cells reached 75-85% confluency, all of the spent media is
aseptically removed from the flasks and discarded. 3m1 of 0.25%
trypsin/EDTA (w/v) solution is added to cover the cell layer, and the
cells are incubated at 37° C., 5% CO2 and 90% humidity for 5
minutes. The flask is tapped once or twice to expedite cell detachment.
Once >95% of the cells are rounded and detached, 7 ml of warm culture
medium is added to each T-75 flask, and the solution is dispersed by
pipetting over the cell layer surface several times.

[0285]After counting the cells and determining viability as above, the
cells are centrifuged at 1000 RPM for 5 minutes at room temperature.
Cells are passaged by gently resuspending the cell pellet from one T-75
flask with culture medium, and evenly plating the cells onto two
FN-coated T-75 flasks.

[0289]Distinct populations of placental cells were obtained from the
placentas of normal, full-term pregnancies. All donors provided full
written consent for the use of their placentas for research purposes.
Placental stem cells were obtained from the following sources: (1)
placental perfusate (from perfusion of the placental vasculature); and
enzymatic digestions of (2) amnion, (3) chorion, (4) amnion-chorion
plate, and (5) umbilical cord. The various placental tissues were cleaned
in sterile PBS (Gibco-Invitrogen Corporation, Carlsbad, Calif.) and
placed on separate sterile Petri dishes. The various tissues were minced
using a sterile surgical scalpel and placed into 50 mL Falcon Conical
tubes. The minced tissues were digested with 1× Collagenase
(Sigma-Aldrich, St. Louis, Mo.) for 20 minutes in a 37° C. water
bath, centrifuged, and then digested with 0.25% Trypsin-EDTA
(Gibco-Invitrogen Corp) for 10 minutes in a 37° C. water bath. The
various tissues were centrifuged after digestion and rinsed once with
sterile PBS (Gibco-Invitrogen Corp). The reconstituted cells were then
filtered twice, once with 100 μm cell strainers and once with 30 μm
separation filters, to remove any residual extracellular matrix or
cellular debris.

[0290]6.2.1.2 Cellular Viability Assessment and Cell Counts

[0291]The manual trypan blue exclusion method was employed post digestion
to calculate cell counts and assess cellular viability. Cells were mixed
with Trypan Blue Dye (Sigma-Aldrich) at a ratio of 1:1, and the cells
were read on hemacytometer.

[0292]6.2.1.3 Cell Surface Marker Characterization

[0293]Cells that were HLA ABC-/CD45-/CD34-/CD133+ were
selected for characterization. Cells having this phenotype were
identified, quantified, and characterized by two of Becton-Dickinson flow
cytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson, San
Jose, Calif., USA). The various placental cells were stained, at a ratio
of about 10 μL of antibody per 1 million cells, for 30 minutes at room
temperature on a shaker. The following anti-human antibodies were used:
Fluorescein Isothiocyanate (FITC) conjugated monoclonal antibodies
against HLA-G (Serotec, Raleigh, N.C.), CD10 (BD Immunocytometry Systems,
San Jose, Calif.), CD44 (BD Biosciences Pharmingen, San Jose, Calif.),
and CD105 (R&D Systems Inc., Minneapolis, Minn.); Phycoerythrin (PE)
conjugated monoclonal antibodies against CD44, CD200, CD117, and CD13 (BD
Biosciences Pharmingen); Phycoerythrin-Cy5 (PE Cy5) conjugated
Streptavidin and monoclonal antibodies against CD 117 (BD Biosciences
Pharmingen); Phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodies
against CD33 and CD10 (BD Biosciences); Allophycocyanin (APC) conjugated
streptavidin and monoclonal antibodies against CD38 (BD Biosciences
Pharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen). After
incubation, the cells were rinsed once to remove unbound antibodies and
were fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at
4° C. The following day, the cells were rinsed twice, filtered
through a 30 μm separation filter, and were run on the flow
cytometer(s).

[0294]Samples that were stained with anti-mouse IgG antibodies (BD
Biosciences Pharmingen) were used as negative controls and were used to
adjust the Photo Multiplier Tubes (PMTs). Samples that were single
stained with anti-human antibodies were used as positive controls and
were used to adjust spectral overlaps/compensations.

[0295]6.2.1.4 Cell Sorting and Culture

[0296]One set of placental cells (from perfusate, amnion, or chorion),
prior to any culture, was stained with 7-Amino-Actinomycin D (7AAD; BD
Biosciences Pharmingen) and monoclonal antibodies specific for the
phenotype of interest. The cells were stained at a ratio of 10 μL of
antibody per 1 million cells, and were incubated for 30 minutes at room
temperature on a shaker. These cells were then positively sorted for live
cells expressing the phenotype of interest on the BD FACS Aria and plated
into culture. Sorted (population of interest) and "All" (non-sorted)
placental cell populations were plated for comparisons. The cells were
plated onto a fibronectin (Sigma-Aldrich) coated 96 well plate at the
cell densities listed in Table 1 (cells/cm2). The cell density, and
whether the cell type was plated in duplicate or triplicate, was
determined and governed by the number of cells expressing the phenotype
of interest.

[0297]Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2%
fetal calf serum (Hyclone Labs.); 1× insulin-transferrin-selenium
(ITS); 1× linoleic acid-bovine serum albumin (LA-BSA); 10-9 M
dexamethasone (Sigma); 10-4 M ascorbic acid 2-phosphate (Sigma);
epidermal growth factor 10 ng/mL (R&D Systems); and platelet-derived
growth factor (PDGF-BB) 10 ng/mL (R&D Systems)) was added to each well of
the 96 well plate and the plate was placed in a 5% CO2/37° C.
incubator. On day 7, 100 μL of complete medium was added to each of
the wells. The 96 well plate was monitored for about two weeks and a
final assessment of the culture was completed on day 12. This is very
early in the placental stem cell culture, and represents passage 0 cells.

[0298]6.2.1.5 Data Analysis

[0299]FACSCalibur data was analyzed in FlowJo (Tree star, Inc) using
standard gating techniques. The BD FACS Aria data was analyzed using the
FACSDiva software (Becton-Dickinson). The FACS Aria data was analyzed
using doublet discrimination gating to minimize doublets, as well as,
standard gating techniques. All results were compiled in Microsoft Excel
and all values, herein, are represented as average±standard deviation
(number, standard error of mean).

[0300]6.2.2 Results

[0301]6.2.2.1 Cellular Viability

[0302]Post-digestion viability was assessed using the manual trypan blue
exclusion method (FIG. 1). The average viability of cells obtained from
the majority of the digested tissue (from amnion, chorion or
amnion-chorion plate) was around 70%. Amnion had an average viability of
74.35%±10.31% (n=6, SEM=4.21), chorion had an average viability of
78.18%±12.65% (n=4, SEM=6.32), amnion-chorion plate had an average
viability of 69.05%±10.80% (n=4, SEM=5.40), and umbilical cord had an
average viability of 63.30%±20.13% (n=4, SEM=10.06). Cells from
perfusion, which did not undergo digestion, retained the highest average
viability, 89.98±6.39% (n=5, SEM=2.86).

[0303]6.2.2.2 Cell Quantification

[0304]The populations of placental cells and umbilical cord cells were
analyzed to determine the numbers of HLA
ABC-/CD45-/CD34-/CD133- cells. From the analysis of
the BD FACSCalibur data, it was observed that the amnion, perfusate, and
chorion contained the greatest total number of these cells,
30.72±21.80 cells (n=4, SEM=10.90), 26.92±22.56 cells (n=3,
SEM=13.02), and 18.39±6.44 cells (n=2, SEM=4.55) respectively (data
not shown). The amnion-chorion plate and umbilical cord contained the
least total number of cells expressing the phenotype of interest,
4.72±4.16 cells (n=3, SEM=2.40) and 3.94±2.58 cells (n=3, SEM=1.49)
respectively (data not shown).

[0305]Similarly, when the percent of total cells expressing the phenotype
of interest was analyzed, it was observed that amnion and placental
perfusate contained the highest percentages of cells expressing this
phenotype (0.0319%±0.0202% (n=4, SEM=0.0101) and 0.0269%±0.0226%
(n=3, SEM=0.0130) respectively (FIG. 2). Although umbilical cord
contained a small number of cells expressing the phenotype of interest
(FIG. 2), it contained the third highest percentage of cells expressing
the phenotype of interest, 0.020±0.0226% (n=3, SEM=0.0131) (FIG. 2).
The chorion and amnion-chorion plate contained the lowest percentages of
cells expressing the phenotype of interest, 0.0184±0.0064% (n=2,
SEM=0.0046) and 0.0177±0.0173% (n=3, SEM=0.010) respectively (FIG. 2).

[0306]Consistent with the results of the BD FACSCalibur analysis, the BD
FACS Aria data also identified amnion, perfusate, and chorion as
providing higher numbers of HLA
ABC-/CD45-/CD34-/CD133+ cells than the remaining
sources. The average total number of cells expressing the phenotype of
interest among amnion, perfusate, and chorion was 126.47±55.61 cells
(n=15, SEM=14.36), 81.65±34.64 cells (n=20, SEM=7.75), and
51.47±32.41 cells (n=15, SEM=8.37), respectively (data not shown). The
amnion-chorion plate and umbilical cord contained the least total number
of cells expressing the phenotype of interest, 44.89±37.43 cells (n=9,
SEM=12.48) and 11.00±4.03 cells (n=9, SEM=1.34) respectively (data not
shown).

[0321]The three distinct populations of placental cells that expressed the
greatest percentages of HLA ABC, CD45, CD34, and CD133 (cells derived
from perfusate, amnion and chorion) were stained with 7AAD and the
antibodies for these markers. The three populations were positively
sorted for live cells expressing the phenotype of interest. The results
of the BD FACS Aria sort are listed in table 2.

[0322]The three distinct populations of positively sorted cells ("sorted")
and their corresponding non-sorted cells were plated and the results of
the culture were assessed on day 12 (Table 3). Sorted perfusate-derived
cells, plated at a cell density of 40,600/cm2, resulted in small,
round, non-adherent cells. Two out of the three sets of non-sorted
perfusate-derived cells, each plated at a cell density of
40,600/cm2, resulted in mostly small, round, non-adherent cells with
several adherent cells located around the periphery of well. Non-sorted
perfusate-derived cells, plated at a cell density of 93,800/cm2,
resulted in mostly small, round, non-adherent cells with several adherent
cells located around the well peripheries.

[0325]Subsequent to the performance of the experiments related above, and
further culture of the placental stem cells, it was determined that the
labeling of the antibodies for CD117 and CD133, in which a
streptavidin-conjugated antibody was labeled with biotin-conjugated
phycoerythrin (PE), produced background significant enough to resemble a
positive reading. This background had initially resulted in the placental
stem cells being deemed to be positive for both markers. When a different
label, APC or PerCP was used, the background was reduced, and the
placental stem cells were correctly determined to be negative for both
CD117 and CD133.

[0327]Placental stem cells or umbilical cord stem cells, obtained by
enzymatic digestion, in culture medium were washed once by adding 2 mL 2%
FBS-PBS and centrifuging at 400 g for 5 minutes. The supernatant was
decanted, and the pellet was resuspended in 100-200 μL 2% FBS-PBS. 4
tubes were prepared with BDTM CompBeads (Cat #552843) by adding 100 μl
of 2% FBS-PBS to each tube, adding 1 full drop (approximately 60 μl)
of the BD® CompBeads Negative Control and 1 drop of the BD®
CompBeads Anti-Mouse beads to each tube, and vortexing. To the 4 tubes of
BD® CompBeads, the following antibodies were added:

[0330]The control and sample tubes were incubated in the dark at room
temperature for 30 minutes. After incubation, the tubes were washed by
adding 2 mL 2% FBS-PBS and centrifuging at 400 g for 5 minutes. The
supernatant was decanted, and the pellet was resuspended in 100-200 μL
2% FBS-PBS and acquire on flow cytometer. All other antibodies were used
following this procedure.

[0332]Flow cytometry results showed that for the placental stem cells that
were tested, 93.83% of cells were CD105+, 90.76% of cells were
CD200+, and 86.93% of cells were both CD105+ and CD200+.
99.97% of cells were CD10+, 99.15% of cells were CD34-, and
99.13% of cells were both CD10+ and CD34-. 98.71% of cells were
cytokeratin positive, 99.95% of cells were CD44+, and 98.71% of
cells were positive for both cytokeratin and CD44. 99.51% of cells were
CD45-, 99.78% of cells were negative for CD133, and 99.39% of cells
were negative for both CD45 and CD133. 99.31% of cells were positive for
CD90, 99.7% were negative for CD117, and 99.01% were positive for CD90
and negative for CD117. 95.7% of cells were negative for both CD80 and
CD86.

[0333]Flow cytometry results for umbilical cord stem cells showed that
95.95% of cells were CD200+, 94.71% were CD105+, and 92.69%
were CD105+ and CD200+. 99.93% of the cells were CD10+,
99.99% of the cells were CD34-, and 99.6% of the cells were both
CD10+ and CD34. 99.45% of the cells were cytokeratin positive,
99.78% of the cells were CD44+, and 99.3% of the cells were positive
for both cytokeratin and CD44. 99.33% of the cells were CD45-,
99.74% were CD133-, and 99.15% of the cells were both CD45- and
CD133-. 99.84% of the cells were CD117-, 98.78% of the cells
were CD90+, and 98.64% of the cells were both CD90- and
CD117-.

[0336]The assay uses ALDEFLUOR®, a fluorescent ALDH substrate
(Aldagen, Inc., Durham, N.C.). The manufacturer's protocol was followed.
The dry ALDEFLUOR® reagent is provided in a stable, inactive form.
The ALDEFLUOR® was activated by dissolving the dry compound in
dimethylsulfoxide (DMSO) and adding 2N HCl, and was added immediately to
the cells. A control tube was also established by combing the cells with
ALDEFLUOR® plus DEAB, a specific inhibitor of ALDH.

[0338]The assay proceeded as follows. Sample concentration was adjusted to
1×106 cells/ml with Assay buffer provided with the
ALDEFLUOR® Assay Kit. 1 mL of adjusted cell suspension into
experimental and control tube for each of the cell lines tested, and 5
μl of DEAB was additionally added to the control tube labeled as
control.

[0339]ALDEFLUOR® substrate was activated by adding 25 μl of DMSO to
the dry ALDEFLUOR® Reagent, and let stand for 1 minute at RT. 25
μl of 2N HCL was added and mixed well. This mixture was incubated for
15 min at RT. 360 μl of ALDEFLUOR® Assay Buffer was added to the
vial and mixed. The resulting mixture was stored at 2-8° C. during
use.

[0340]5 μl of the activated ALDEFLUOR® reagent was added per 1
milliliter of sample to the experimental tubes, and 0.5 ml of this
mixture was immediately transferred into the control tubes. The
experimental and control tubes for each cell line were incubated for 30
minutes at 37° C. After incubation, the tubes were centrifuged at
400×g, and the supernatant was discarded. The cells in the
resulting pellet were resuspended in 0.5 ml Assay Buffer and analyze by
flow cytometry. Data was analyzed using FLOWJO® software (Tree Star,
Ashland, Oreg.). SSC vs FSC and SSC vs FL1 plots were created in the
FLOWJO® workspace. Control and experimental data files were opened for
each sample, and the appropriate gates were determined based on control
samples. Positive cells were calculated as a percent ALDEFLUOR®
positive out of the total number of events counted.

[0341]Placental stem cell lines demonstrated ALDH activity of from about
3% to about 25% (3.53%, 8.76% and 25.26%). Umbilical cord stem cell lines
demonstrated ALDH activity of from about 16% to about 20% (16.59%,
17.01%, 18.44% and 19.83%). In contrast, BM-MSC and HVT were negative and
1.5% respectively for ALDH, but the adipose derived MSC is close to 30%
ALDH+. The positive control CD34+ cells purified from umbilical
cord blood were, as expected, highly positive (75%) for ALDH.

[0343]A post-partum placenta is obtained within 24 hours after birth. The
umbilical cord is clamped with an umbilical cord clamp approximately 3 to
4 inches about the placental disk, and the cord is cut above the clamp.
The umbilical cord is either discarded, or processed to recover, e.g.,
umbilical cord stem cells, and/or to process the umbilical cord membrane
for the production of a biomaterial. Excess amniotic membrane and chorion
is cut from the placenta, leaving approximately 1/4 inch around the edge
of the placenta. The trimmed material is discarded.

[0344]Starting from the edge of the placental membrane, the amniotic
membrane is separated from the chorion using blunt dissection with the
fingers. When the amniotic membrane is entirely separated from the
chorion, the amniotic membrane is cut around the base of the umbilical
cord with scissors, and detached from the placental disk. The amniotic
membrane can be discarded, or processed, e.g., to obtain stem cells by
enzymatic digestion, or to produce, e.g., an amniotic membrane
biomaterial.

[0345]The fetal side of the remaining placental material is cleaned of all
visible blood clots and residual blood using sterile gauze, and is then
sterilized by wiping with an iodine swab than with an alcohol swab. The
umbilical cord is then clamped crosswise with a sterile hemostat beneath
the umbilical cord clamp, and the hemostat is rotated away, pulling the
cord over the clamp to create a fold. The cord is then partially cut
below the hemostat to expose a cross-section of the cord supported by the
clamp. Alternatively, the cord is clamped with a sterile hemostat. The
cord is then placed on sterile gauze and held with the hemostat to
provide tension. The cord is then cut straight across directly below the
hemostat, and the edge of the cord near the vessel is re-clamped.

[0346]The vessels exposed as described above, usually a vein and two
arteries, are identified, and opened as follows. A closed alligator clamp
is advanced through the cut end of each vessel, taking care not to
puncture the clamp through the vessel wall. Insertion is halted when the
tip of the clamp is slightly above the base of the umbilical cord. The
clamp is then slightly opened, and slowly withdrawn from the vessel to
dilate the vessel.

[0347]Plastic tubing, connected to a perfusion device or peristaltic pump,
is inserted into each of the placental arteries. Plastic tubing,
connected to a 250 mL collection bag, is inserted into the placental
vein. The tubing is taped into place.

[0348]A small volume of sterile injection grade 0.9% NaCl solution to
check for leaks. If no leaks are present, the pump speed is increased,
and about 750 mL of the injection grade 0.9% NaCl solution is pumped
through the placental vasculature. Perfusion can be aided by gently
massaging the placental disk from the outer edges to the cord. When a
collection bag is full, the bag is removed from the coupler connecting
the tubing to the bag, and a new bag is connected to the tube.

[0349]When collection is finished, the collection bags are weighed and
balanced for centrifugation. After centrifugation, each bag is placed
inside a plasma extractor without disturbing the pellet of cells. The
supernatant within the bags is then removed and discarded. The bag is
then gently massaged to resuspend the cells in the remaining supernatant.
Using a sterile 1 mL syringe, about 300-500 μL of cells is withdrawn
from the collection bag, via a sampling site coupler, and transferred to
a 1.5 mL centrifuge tube. The weight and volume of the remaining
perfusate are determined, and 1/3 volume of hetastarch is added to the
perfusate and mixed thoroughly. The number of cells per mL is determined.
Red blood cells are removed from the perfusate using a plasma extractor.

[0350]Placental cells are then immediately cultured to isolate placental
stem cells, or are cryopreserved for later use.

6.6 Example 6

Differentiation of Placental Stem Cells

6.6.1 Induction of Differentiation into Neurons

[0351]Neuronal differentiation of placental stem cells can also be
accomplished as follows: [0352]1. Placental stem cells are grown for 24
hr in preinduction medium consisting of DMEM/20% FBS and 1 mM
beta-mercaptoethanol. [0353]2. The preinduction medium is removed and
cells are washed with PBS. [0354]3. Neuronal induction medium consisting
of DMEM and 1-10 mM betamercaptoethanol is added to the cells.
Alternatively, induction media consisting of DMEM/2% DMSO/200 μM
butylated hydroxyanisole may be used. [0355]4. In certain embodiments,
morphologic and molecular changes may occur as early as 60 minutes after
exposure to serum-free media and betamercaptoethanol. RT/PCR may be used
to assess the expression of e.g., nerve growth factor receptor and
neurofilament heavy chain genes.

6.6.2 Induction of Differentiation into Adipocytes

[0356]Several cultures of placental stem cells derived from enzymatic
digestion of amnion, at 50-70% confluency, were induced in medium
comprising (1) DMEM/MCDB-201 with 2% FCS, 0.5% hydrocortisone, 0.5 mM
isobutylmethylxanthine (IBMX), 60 μM indomethacin; or (2)
DMEM/MCDB-201 with 2% FCS and 0.5% linoleic acid. Cells were examined for
morphological changes; after 3-7 days, oil droplets appeared.
Differentiation was also assessed by quantitative real-time PCR to
examine the expression of specific genes associated with adipogenesis,
i.e., PPAR-γ2, aP-2, lipoprotein lipase, and osteopontin. Two
cultures of placental stem cells showed an increase of 6.5-fold and
24.3-fold in the expression of adipocyte-specific genes, respectively.
Four other cultures showed a moderate increase (1.5-2.0-fold) in the
expression of PPAR-γ2 after induction of adipogenesis.

[0357]In another experiment, placental stem cells obtained from perfusate
were cultured in DMEM/MCDB-201 (Chick fibroblast basal medium) with 2%
FCS. The cells were trypsinized and centrifuged. The cells were
resuspended in adipo-induction medium (AIM) 1 or 2. AIM1 comprised
MesenCult Basal Medium for human Mesenchymal Stem Cells (StemCell
Technologies) supplemented with Mesenchymal Stem Cell Adipogenic
Supplements (StemCell Technologies). AIM2 comprised DMEM/MCDB-201 with 2%
FCS and LA-BSA (1%). About 1.25×105 placental stem cells were
grown in 5 mL AIM1 or AIM2 in T-25 flasks. The cells were cultured in
incubators for 7-21 days. The cells developed oil droplet vacuoles in the
cytoplasm, as confirmed by oil-red staining, suggesting the
differentiation of the stem cells into adipocytes.

[0358]Adipogenic differentiation of placental stem cells can also be
accomplished as follows: [0359]1. Placental stem cells are grown in
MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum. [0360]2.
Three cycles of induction/maintenance are used. Each cycle consists of
feeding the placental stem cells with Adipogenesis Induction Medium
(Cambrex) and culturing the cells for 3 days (at 37° C., 5%
CO2), followed by 1-3 days of culture in Adipogenesis Maintenance
Medium (Cambrex). An alternate induction medium that can be used contains
1 μM dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5 mM
IBMX, DMEM-high glucose, FBS, and antibiotics. [0361]3. After 3 complete
cycles of induction/maintenance, the cells are cultured for an additional
7 days in adipogenesis maintenance medium, replacing the medium every 2-3
days. [0362]4. A hallmark of adipogenesis is the development of multiple
intracytoplasmic lipid vesicles that can be easily observed using the
lipophilic stain oil red O. Expression of lipase and/or fatty acid
binding protein genes is confirmed by RT/PCR in placental stem cells that
have begun to differentiate into adipocytes.

[0363]6.6.3 Induction of Differentiation into Osteocytes

[0364]Osteogenic medium was prepared from 185 mL Cambrex Differentiation
Basal Medium--Osteogenic and SingleQuots (one each of dexamethasone,
1-glutamine, ascorbate, pen/strep, MCGS, and β-glycerophosphate).
Placental stem cells from perfusate were plated, at about
3×103 cells per cm2 of tissue culture surface area in
0.2-0.3 mL MSCGM per cm2 tissue culture area. Typically, all cells
adhered to the culture surface for 4-24 hours in MSCGM at 37° C.
in 5% CO2. Osteogenic differentiation was induced by replacing the
medium with Osteogenic Differentiation medium. Cell morphology began to
change from the typical spindle-shaped appearance of the adherent
placental stem cells, to a cuboidal appearance, accompanied by
mineralization. Some cells delaminated from the tissue culture surface
during differentiation.

[0365]Osteogenic differentiation can also be accomplished as follows:
[0366]1. Adherent cultures of placental stem cells are cultured in MSCGM
(Cambrex) or DMEM supplemented with 15% cord blood serum. [0367]2.
Cultures are cultured for 24 hours in tissue culture flasks. [0368]3.
Osteogenic differentiation is induced by replacing MSCGM with Osteogenic

[0399]The Example demonstrates the differentiation of placental stem cells
into chondrogenic cells and the development of cartilage-like tissue from
such cells.

[0400]Cartilage is an avascular, alymphatic tissue that lacks a nerve
supply. Cartilage has a low chondrocyte density (<5%), however these
cells are surprisingly efficient at maintaining the extracellular matrix
around them. Three main types of cartilage exist in the body: (1)
articular cartilage, which facilitates joint lubrication in joints; (2)
fibrocartilage, which provides shock absorption in, e.g., meniscus and
intervertebral disc; and (3) elastic cartilage, which provides anatomical
structure in, e.g., nose and ears. All three types of cartilage are
similar in biochemical structure.

[0401]Joint pain is a major cause of disability and provides an unmet need
of relief in the area of orthopedics. Primary osteoarthritis (which can
cause joint degeneration), and trauma are two common causes of pain.
Approximately 9% of the U.S. population has osteoarthritis of hip or
knee, and more than 2 million knee surgeries are performed yearly.
Unfortunately, current treatments are more geared towards treatment of
symptoms rather than repairing the cartilage. Natural repair occurs when
fibroblast-like cells invade the area and fill it with fibrous tissue
which is neither as resilient or elastic as the normal tissue, hence
causing more damage. Treatment options historically included tissue
grafts, subchondral drilling, or total joint replacement. More recent
treatments however include CARTICEL®, an autologous chondrocyte
injection; SYNVISC® and ORTHOVISC®, which are hyaluronic acid
injections for temporary pain relief; and CHONDROGEN®, an injection of
adult mesenchymal stem cells for meniscus repair. In general, the trend
seems to be lying more towards cellular therapies and/or tissue
engineered products involving chondrocytes or stem cells.

[0403]Placental and umbilical cord stem cells were isolated and purified
from full term human placenta by enzymatic digestion. Human MSC cells and
HDF cells were purchased from Cambrex, and MC3T3 cells were purchased
from American Type Culture Collection. All cell lines used were
centrifuged into pellets in polypropylene centrifuge tubes at 800 RPM for
5 minutes and grown in both chondrogenic induction media (Cambrex) and
non-inducing basal MSC media (Cambrex). Pellets were harvested and
histologically analyzed at 7, 14, 21 and 28 days by staining for
glycosaminoglycans (GAGs) with Alcian Blue, and/or for collagens with
Sirius Red. Collagen type was further assessed with immunostaining RNA
analysis for cartilage-specific genes was performed at 7 and 14 days.

[0404]Results

[0405]Experiment 1: Chondrogenesis studies were designed to achieve three
main objectives: (1) to demonstrate that placental and umbilical cord
stem cells can differentiate and form cartilage tissue; (2) to
demonstrate that placental and umbilical cord stem cells can
differentiate functionally into chondrocytes; and (3) to validate results
obtained with the stem cells by evaluating control cell lines.

[0406]For objective 1, in a preliminary study, one placental stem cell
line was cultured in chondrogenic induction medium in the form of cell
pellets, either with or without bone morphogenic protein (BMP) at a final
concentration of 500 ng/mL. Pellets were assessed for evidence of
chondrogenic induction every week for 4 weeks. Results indicated that the
pellets do increase in size over time. However, no visual differences
were noted between the BMP- and BMP- samples. Pellets were also
histologically analyzed for GAG's, an indicator of cartilage tissue, by
staining with Alcian Blue. BMP+ cells generally appeared more
metabolically active with pale vacuoles whereas BMP- cells were
smaller with dense-stained nuclei and less cytoplasm (reflects low
metabolic activity). At 7 days, BMP+ cells had stained heavily blue,
while BMP- had stained only faintly. By 28 days of induction, both
BMP- and BMP- cells were roughly equivalently stained with
Alcian Blue. Overall, cell density decreased over time, and matrix
overtook the pellet. In contrast, the MC3T3 negative cell line did not
demonstrate any presence of GAG when stained with Alcian Blue.

[0407]Experiment 2: Based on the results of Experiment 1, a more detailed
study was designed to assess the chondrogenic differentiation potential
of two placental stem cell and two umbilical cord stem cell lines. In
addition to the Alcian Blue histology, cells were also stained with
Sirius Red, which is specific for type II collagen. Multiple pellets were
made for each cell line, with and without induction media.

[0408]The pelleted, cultured cell lines were first assessed by gross
observation for macroscopic generation of cartilage. Overall, the stem
cell lines were observed to make pellets as early as day 1. These pellets
grew over time and formed a tough matrix, appearing white, shining and
cartilage-like, and became mechanically tough. By visual inspection,
pellets from placental stem cells or umbilical cord stem cells were much
larger than the MSC controls. Control pellets in non-induction media
started to fall apart by Day 11, and were much smaller at 28 days than
pellets developed by cells cultured in chondrogenic induction medium.
Visually, there were no differences between pellets formed by placental
stem cells or umbilical cord. However, the UC67249 stem cell line, which
was initiated in dexamethasone-free media, formed larger pellets.
Negative control MC3T3 cells did not form pellets; however, HDFs did form
pellets.

[0409]Representative pellets from all test groups were then subjected to
histological analysis for GAG's and collagen. Generally, pellets formed
by the stem cells under inducing conditions were much larger and stayed
intact better than pellets formed under non-inducing conditions. Pellets
formed under inducing conditions showed production of GAGs and increasing
collagen content over time, and as early as seven days, while pellets
formed under non-inducing conditions showed little to no collagen
production, as evidenced by weak Alcian Blue staining In general, the
placental stem cells and umbilical cord stem cells appeared, by visual
inspection, to produce tougher, larger pellets, and appeared to be
producing more collagen over time, than the hMSCs. Moreover, over the
course of the study, the collagen appeared to thicken, and the collagen
type appeared to change, as evidenced by changes in the fiber colors
under polarized light (colors correlate to fiber thickness which may be
indicative of collagen type). Non-induced placental stem cells produced
much less type II collagen, if any, compared to the induced stem cells.
Over the 28-day period, cell density decreased as matrix production
increased, a characteristic of cartilage tissue.

[0410]These studies confirm that placental and umbilical cord stem cells
can be differentiated along a chondrogenic pathway, and can easily be
induced to form cartilage tissue. Initial observations indicate that such
stem cells are preferable to MSCs for the formation of cartilage tissue.

6.7 Example 7

Hanging Drop Culture of Placental Stem Cells

[0411]Placental adherent stem cells in culture are trypsinized at
37° C. for about 5 minutes, and loosened from the culture dish by
tapping. 10% FBS is added to the culture to stop trypsinization. The
cells are diluted to about 1×104 cells per mL in about 5 mL of
medium. Drops (either a single drop or drops from a multi-channel
micropipette are placed on the inside of the lid of a 100 mL Petri dish.
The lid is carefully inverted and placed on top of the bottom of the
dish, which contains about 25 ml of sterile PBS to maintain the moisture
content in the dish atmosphere. Cells are grown for 6-7 days.

[0413]Approximately 10 grams of placental tissue (amnion and chorion) is
obtained, macerated, and digested using equal volumes of collagenase A (1
mg/ml) (Sigma) and Trypsin-EDTA (0.25%) (Gibco-BRL) in a total volume of
about 30 ml for about 30 minutes at 37° C. Cells liberated by the
digestion are washed 3× with culture medium, distributed into four
T-225 flasks and cultured as described in Example 1. Placental stem cell
yield is between about 4×108 and 5×108 cells per 10
g starting material. Cells, characterized at passage 3, are predominantly
CD10+, CD90+, CD105+, CD200+, CD34- and
CD45-.

6.9 Example 9

Production of Cryopreserved Stem Cell Product and Stem Cell Bank

[0414]This Example demonstrates the isolation of placental stem cell and
the production of a frozen stem cell-based product.

[0415]Summary: Placental tissue is dissected and digested, followed by
primary and expansion cultures to achieve an expanded cell product that
produces many cell doses. Cells are stored in a two-tiered cell bank and
are distributed as a frozen cell product. All cell doses derived from a
single donor placenta are defined as a lot, and one placenta lot is
processed at a time using sterile technique in a dedicated room and Class
100 laminar flow hood. The cell product is defined as being CD105+,
CD200+, CD10+, and CD34-, having a normal karyotype and no
or substantially no maternal cell content.

6.9.1 Obtaining Stem Cells

[0416]Tissue Dissection and Digestion: A placenta is obtained less than 24
hours after expulsion. Placental tissue is obtained from amnion, a
combination of amnion and chorion, or chorion. The tissue is minced into
small pieces, about 1 mm in size. Minced tissue is digested in 1 mg/ml
Collagenase 1A for 1 hour at 37° C. followed by Trypsin-EDTA for
30 minutes at 37° C. After three washes in 5% FBS in PBS, the
tissue is resuspended in culture medium.

[0417]Primary Culture: The purpose of primary culture is to establish
cells from digested placental tissue. The digested tissue is suspended in
culture medium and placed into Corning T-flasks, which are incubated in a
humidified chamber maintained at 37° C. with 5% CO2. Half of
the medium is replenished after 5 days of culture. High-density colonies
of cells form by 2 weeks of culture. Colonies are harvested with
Trypsin-EDTA, which is then quenched with 2% FBS in PBS. Cells are
centrifuged and resuspended in culture medium for seeding expansion
cultures. These cells are defined as Passage 0 cells having doubled 0
times.

[0418]Expansion Culture: Cells harvested from primary culture, harvested
from expansion culture, or thawed from the cell bank are used to seed
expansion cultures. Cell Factories (NUNC®) are treated with 5%
CO2 in air at 50 ml/min/tray for 10 min through a sterile filter and
warmed in a humidified incubator maintained at 37° C. with 5%
CO2. Cell seeds are counted on a hemacytometer with trypan blue, and
cell number, viability, passage number, and the cumulative number of
doublings are recorded. Cells are suspended in culture medium to about
2.3×104 cells/ml and 110 ml/tray are seeded in the Cell
Factories. After 3-4 days and again at 5-6 days of culture, culture
medium is removed and replaced with fresh medium, followed by another
treatment with 5% CO2 in air. When cells reach approximately
105 cells/cm2, cells are harvested with Trypsin-EDTA, followed
by quenching with 2% FBS in PBS. Cell are then centrifuged and
resuspended in culture medium.

[0419]Cryopreservation: Cells to be frozen down are harvested from culture
with Trypsin-EDTA, quenched with 2% FBS in PBS, and counted on a
hemacytometer. After centrifugation, cells are resuspended with 10% DMSO
in FBS to a concentration of about 1 million cells/ml for cells to be
used for assembly of a cell bank, and 10 million cells/ml for individual
frozen cell doses. The cell solution is transferred to a freezing
container, which is placed in an isopropyl alcohol bath in a -80°
C. freezer. The following day, cells are transferred to liquid nitrogen.

[0420]6.9.2 Design of a Stem Cell Bank

[0421]A "lot" is defined as all cell doses derived from a single donor
placenta. Cells maintained normal growth, karyotype, and cell surface
maker phenotype for over 8 passages and 30 doublings during expansion
culture. Given this limitation, doses comprise cells from 5 passages and
about 20 doublings. To generate a supply of equivalent cells, a single
lot is expanded in culture and is stored in a two-tiered cell bank and
frozen doses. In particular, cells harvested from the primary culture,
which are defined as Passage 0 cells having undergone 0 doublings, are
used to initiate an expansion culture. After the first passage,
approximately 4 doublings occur, and cells are frozen in a Master Cell
Bank (MCB). Vials from the MCB are used to seed additional expansion
cultures. After two additional passages of cells thawed from the MCB,
cells are frozen down in a Working Cell Bank (WCB), approximately 12
cumulative doublings. Vials from the WCB are used to seed an expansion
culture for another 2 passages, resulting in Passage 5 cells at
approximately 20 doublings that are frozen down into individual doses.

[0422]6.9.3 Thawing Cells for Culture

[0423]Frozen containers of cells are placed into a sealed plastic bag and
immersed in a 37° C. water bath. Containers are gently swirled
until all of the contents are melted except for a small piece of ice.
Containers are removed from the sealed plastic bag and a 10× volume
of culture medium is slowly added to the cells with gentle mixing. A
sample is counted on the hemacytometer and seeded into expansion
cultures.

[0424]6.9.4 Thawing Cells for Injection

[0425]Frozen containers of cells are transferred to the administration
site in a dry nitrogen shipper. Prior to administration, containers are
placed into a sealed plastic bag and immersed in a 37° C. water
bath. Containers are gently swirled until all of the contents are melted
except for a small piece of ice. Containers are removed from the sealed
plastic bag and an equal volume of 2.5% HSA/5% Dextran is added. Cells
are injected with no further washing.

[0426]6.9.5 Testing and Specifications

[0427]A maternal blood sample accompanies all donor placentas. The sample
is screened for Hepatitis B core antibody and surface antigen, Hepatitis
C Virus antibody and nucleic acid, and HIV I and II antibody and nucleic
acid. Placental processing and primary culture begins prior to the
receipt of test results, but continues only for placentas associated with
maternal blood samples testing negative for all viruses. A lot is
rejected if the donor tests positive for any pathogen. In addition, the
tests described in Table 3 are performed on the MCB, the WCB, and a
sample of the cell dose material derived from a vial of the WCB. A lot is
released only when all specifications are met.

[0429]Cells are placed in 1% paraformaldehyde (PFA) in PBS for 20 minutes
and stored in a refrigerator until stained (up to a week). Cells are
washed with 2% FBS, 0.05% sodium azide in PBS (Staining Buffer) and then
resuspended in staining buffer. Cells are stained with the following
antibody conjugates: CD105-FITC, CD200-PE, CD34-PECy7, CD10-APC. Cells
are also stained with isotype controls. After 30 minute incubation, the
cells are washed and resuspended with Staining Buffer, followed by
analysis on a flow cytometer. Cells having an increased fluorescence
compared to isotype controls are counted as positive for a marker.

6.10 Example 10

Identification of Placental Stem Cell-Specific Genes

[0430]Gene expression patterns from placental stem cells from
amnion-chorion (AC) and umbilical cord (UC) were compared to gene
expression patterns of multipotent bone marrow-derived mesenchymal stem
cells (BM) and dermal fibroblasts (DF), the latter of which is considered
to be terminally differentiated. Cells were grown for a single passage,
an intermediate number of passages, and large number of passages
(including until senescence). Results indicate that the number of
population doublings has a major impact on gene expression. A set of
genes was identified that are up-regulated in AC and UC, and either
down-regulated or absent in BM and DF, and that are expressed independent
of passage number. This set of placental stem cell- or umbilical cord
stem cell-specific genes encodes a number of cytoskeleton and
cell-to-cell adhesion proteins associated with epithelial cells and an
immunoglobulin-like surface protein, CD200, implicated in maternal-fetal
immune tolerance. Placental stem cells and umbilical cord stem cells will
be referred to collectively hereinafter in this Example as AC/UC stem
cells.

[0431]6.10.1 Methods and Materials

[0432]6.10.1.1 Cells and Cell Culture

[0433]BM (Cat #PT-2501) and DF (Cat #CC-2511) were purchased from Cambrex.
AC and UC originated from passage 0 tissue culture flasks. AC and UC in
the flasks were obtained by digestion from a donor placenta designated
2063919. T-75 culture flasks were seeded at 6000 cells/cm2 and cells
were passaged when they became confluent. Population doublings were
estimated from trypan blue cell counts. Cultures were assayed for gene
expression after 3, 11-14, and 24-38 population doublings.

[0434]6.10.1.2 RNA, Microarrays, and Analysis

[0435]Cells were lysed directly in their tissue culture flasks, with the
exception of one culture that was trypsinized prior to lysis. Total RNA
was isolated with the RNeasy kit from QIAGEN. RNA integrity and
concentrations were determined with an Agilent 2100 Bioanalyzer. Ten
micrograms of total RNA from each culture were hybridized on an
Affymetrix GENECHIP® platform. Total RNA was converted to labeled
cRNAs and hybridized to oligonucleotide Human Genome U133A 2.0 arrays
according to the manufacture's methods. Image files were processed with
the Affymetrix MAS 5.0 software, and normalized and analyzed with Agilent
GeneSpring 7.3 software.

[0438]To establish a gene expression pattern unique to AC/UC stem cells,
two stem cell lines, AC(6) and UC(6), were cultured in parallel with
BM-MSC and DF. To maximize identifying a gene expression profile
attributable to cellular origin and minimize exogenous influences all
cells were grown in the same medium, seeded, and sub-cultured using the
same criteria. Cells were harvested after 3 population doublings, 11-14
doublings, or 35 doublings or senescence, whichever came first. Genes
whose expression in AC/UC stem cells are unchanged by time-in-culture and
are up-regulated relative to BM and DF are candidates for AC/UC stem
cell-specific genes.

[0439]FIG. 10 shows growth profiles for the four cell lines in the study;
circles indicate which cultures were harvested for RNA isolation. In
total twelve samples were collected. BM, AC(6), and UC(6) were harvested
after three population doublings; these samples were regarded as being in
culture for a "short" period of time. A short-term DF sample was not
collected. Intermediate length cultures, 11 to 14 doublings, were
collected for all cell types. Long-term cultures were collected from all
cell lines at about 35 population doublings or just prior to senescence,
whichever came first. Senescence occurred before 15 doublings for BM and
at 25 doublings for DF. The purchased BM and DF cells were expanded many
times prior to gene analysis, and cannot be considered early-stage.
However, operationally, BM grown for three doublings (BM-03) are deemed a
short-term culture. Likewise, BM-11 is operationally referred to as an
intermediate length culture, but because senescence occurred at 14
doublings, BM-11 is most likely a long-term culture biologically.

[0441]Microarray analysis identifies patterns of gene expression, and
hierarchical clustering (HC) attempts to find similarities in the context
of two dimensions--genes in the first dimension and different conditions
(different RNA samples) in the second. The GeneChips used in this
experiment contained over 22,000 probe sets (referred to as the "all
genes list"), but many of these sets interrogate genes that are not
expressed in any condition. To reduce the all genes list, genes not
expressed or expressed at low levels (raw values below 250) in all
samples were eliminated to yield a list of 8,215 genes.

[0442]6.10.2.3 Gene Expression Analysis Using the Line Graph View

[0443]Gene expression patterns of the 8215 genes were displayed using the
line graph view in GeneSpring (FIG. 11). The x-axis shows the twelve
experimental conditions and the y-axis shows the normalized probe set
expression values on a log scale. The y-axis covers a 10,000-fold range,
and genes that are not expressed or expressed at very low levels are set
to a value of 0.01. By default the normalized value is set to 1. Each
line represents a single gene (actually a probe set, some genes have
multiple probe sets) and runs across all twelve conditions as a single
color. Colors depict relative expression levels, as described for the
heatmaps, but the coloring pattern is determined by selecting one
condition. AC-03 is the selected condition in FIG. 11. Genes up-regulated
relative to the normalized value are displayed by the software as red,
and those that are down-regulated, are displayed as blue. The obvious
upward and downward pointing spikes in AC-03 through UC-11 indicate that
many genes are differentially expressed across these conditions. The
striking similarity in the color patterns between AC-03 and UC-03 show
that many of the same genes are up or down-regulated in these two
samples. Horizontal line segments indicate that a gene's expression level
is unchanged across a number of conditions. This is most notable by
comparing UC-36, UC-38, and UC-38-T. There are no obvious spikes, but
there is a subtle trend in that a number of red lines between UC-36 and
UC-38-T are below the normalized value of 1. This indicates that these
genes, which are up-regulated in AC-03 and UC-03, are down-regulated in
the later cultures. The fact that the expression patterns between UC-38
and UC-38-T are so similar indicates that trypsinizing cells just prior
to RNA isolation has little effect on gene expression.

[0444]In addition to the computationally intensive HC method, by visual
inspection the two BM samples are more similar to each other than to the
other conditions. The same is true for the two DF cultures. And despite
the large number of differentially expressed genes present in the BM and
DF samples, the general appearance suggests that two BMs and the two DFs
are more similar to each other than to AC/UC stem cells. This is
confirmed by the HC results described above.

[0445]When the above process is applied using AC-11 as the selected
condition, it is clear that AC-11 and UC-11 share many of the same
differentially expressed genes, but the total number of genes in common
between these two conditions appears less than the number of
differentially expressed genes shared by AC-03 and UC-03. FIG. 12 shows
genes differentially over-expressed, by six-fold or more relative to the
baseline, in AC-03. The majority of genes up-regulated in AC-03 are also
up-regulated in UC-03, and more divergent in BM and DF.

[0447]Genes that remain constant across all AC/UC samples, and are
down-regulated in BM and DF, are considered AC/UC stem cell-specific. Two
filtering methods were combined to create a list of 58 AC/UC stem
cell-specific genes (Table 4).

[0448]First, 58 genes were identified by selecting those genes
over-expressed≧three-fold in at least seven of eight AC/UC stem
cell conditions relative to all BM and DF samples (FIG. 13). Filtering on
eight of the eight AC/UC stem cell conditions yielded a similar list. The
second filtering method used "absent" and "present" calls provided by the
Affymetrix MAS 5.0 software. A list was created by identifying genes
absent in all BM and DF conditions and present in AC-03, AC-11, UC-03,
and UC-11. Gene calls in the later AC/UC stem cell conditions were not
stipulated.

[0449]The two lists overlapped significantly and were combined. The
combined list was trimmed further by eliminating (1) several genes
expressed at very low levels in most or all AC/UC stem cell conditions,
and (2) genes carried on the Y chromosome. AC and UC cells used in this
study were confirmed to be male by FISH analysis, and the BM and DF were
derived from a female donor. The resulting list of 46 AC/UC stem
cell-specific genes is shown in Table 5.

[0450]This list of 46 genes encodes a collection of proteins presenting a
number of ontology groups. The most highly represented group, cell
adhesion, contains eight genes. No genes encode proteins involved in DNA
replication or cell division. Sixteen genes with specific references to
epithelia are also listed.

[0451]6.10.3 Discussion

[0452]An expression pattern specific to placental stem cells, and
distinguishable from bone marrow-derived mesenchymal cells, was
identified. Operationally, this pattern includes 46 genes that are over
expressed in all placental stem cell samples relative to all BM and DF
samples.

[0453]The experimental design compared cells cultured for short, medium,
and long periods of time in culture. For AC and UC cells, each culture
period has a characteristic set of differentially expressed genes. During
the short-term or early phase (AC-03 and UC-03) two hundred up-regulated
genes regress to the mean after eight population doublings. Without being
bound by theory, it is likely that this early stage gene expression
pattern resembles the expression profile of AC and UC while in the
natural placental environment. In the placenta these cells are not
actively dividing, they are metabolizing nutrients, signaling between
themselves, and securing their location by remodeling the extracellular
surroundings.

[0454]Gene expression by the intermediate length cultures is defined by
rapid cell division and genes differentially expressed at this time are
quite different from those differentially expressed during the early
phase. Many of the genes up-regulated in AC-11 and UC-11, along with
BM-03 and DF-14, are involved in chromosome replication and cell
division. Based on gene expression, BM-03 appears biologically to be a
mid-term culture. In this middle stage cell type-specific gene expression
is overshadowed by cellular proliferation. In addition, almost every gene
over expressed in the short-term AC or UC cultures is down-regulated in
the middle and later stage conditions. 143 genes were up-regulated
five-fold during this highly proliferative phase, constituting
approximately 1.7% of the expressed genes.

[0455]The long-term cultures represent the final or senescent phase. In
this phase, cells have exhausted their ability to divide, and, especially
for AC and UC, the absolute number of differentially expressed genes is
noticeably reduced. This may be the result of cells being fully adapted
to their culture environment and a consequently reduced burden to
biosynthesize. Surprisingly, late BM and DF cultures do not display this
same behavior; a large number of genes are differentially expressed in
BM-11 and DF-24 relative to AC and UC and the normalized value of 1. AC
and UC are distinguishable from BM and DF most notably in the long-term
cultures.

[0456]The placental stem cell-specific gene list described here is
diverse. COL4A1 and COL4A2 are coordinately regulated, and KRT18 and KRT8
also appear to be co-expressed. Eight of the genes encode proteins
involved in cell to cell contact, three of which (DSC3, DSG2, and PKP2)
are localized to desmosomes, intercellular contact points anchored to
intermediate filament cytoskeleton proteins such as keratin 18 and
keratin 8. Tight cell-to-cell contact is characteristic of epithelial and
endothelial cells and not typically associated with fibroblasts. Table 3
lists 16 genes, of the 46 total, characteristic to epithelial cells.
Placental stem cells are generally described as fibroblast-like small
spindle-shaped cells. This morphology is typically distinct from BM and
DF, especially at lower cell densities. Also of note is the expression
pattern of CD200, which is present in AC/UC stem cell and absent in all
BM and DF samples. Moreover, CD200 has been shown to be associated with
immune tolerance in the placenta during fetal development (see, e.g.,
Clark et al., Am. J. Reprod. Immunol. 50(3):187-195 (2003)).

[0457]This subset of genes of 46 genes constitutes a set of molecular
biomarkers that distinguishes AC/UC stem cells from bone marrow-derived
mesenchymal stem cells or fibroblasts.